Human N-type calcium channel isoform and uses thereof

ABSTRACT

The invention pertains to a human N-type calcium channel isoform, hα 1B+SFVG , which is involved in central nervous system signaling, and nucleic acids relating thereto. The present invention also includes fragments and biologically functional variants of the human hα 1B+sFvG channel. Also included are human N-type calcium channel hα 1B+SFVG  subunit inhibitors which inhibit human N-type calcium channel hα 1B+SFVG  subunit activity by inhibiting the expression or function of human N-type calcium channel hα 1B+SFVG  subunit. The invention further relates to methods of using such nucleic acids, polypeptides, and inhibitors in the treatment and/or diagnosis of disease, such as in methods for treating stroke, pain, e.g., neuropathic pain, and traumatic brain injury.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/268,163, filed Mar. 12, 1999, now pending, and claims the benefitunder 35 U.S.C. § 119(e) of U.S. provisional application serial No.60/077,901, filed Mar. 13, 1998, the disclosures of which areincorporated by reference herein.

FIELD OF THE INVENTION

[0002] The invention pertains to human N-type calcium channel α_(1B)subunit isoforms.

BACKGROUND OF THE INVENTION

[0003] Voltage gated calcium channels, also known as voltage dependentcalcium channels (VDCCs) are multisubunit membrane spanning proteinswhich permit controlled calcium influx from an extracellular environmentinto the interior of a cell. Several types of voltage gated calciumchannel have been described in different tissues, including N-type,P/Q-type, L-type and T-type channels. A voltage gated calcium channelpermits entry into the cell of calcium upon depolarization of themembrane of the cell, which is a lessening of the difference inelectrical potential between the outside and the inside of the cell.

[0004] A voltage gated calcium channel contains several proteins,including all (α₁, α₂, β, and γ subunits. Subtypes of the calciumchannel subunits also are known. For instance, α₁ subtypes includeα_(1A), α_(1B), α_(1C), α_(1D), α_(1E) and α_(1S). Each subunit may haveone or more isoforms which result from alternative splicing of RNA inthe formation of a completed messenger RNA which encodes the subunit.For example, at least four isoforms of the rat N-type α_(1B) subunit areknown (see, e.g., Lin et al., Neuron 18:153-166, 1997).

[0005] Isoforms of calcium channel al subunits may be expresseddifferently in different tissues (see, e.g., Lin et al., 1997).Differential expression of subunits isoforms raises the possibility ofdeveloping therapeutics which are specific for distinct isoforms of theα₁, subunits, thereby lessening side effects resulting from the use oftherapeutics which are effective for more than one calcium channelisoform. Two isoforms of the human N-type calcium channel α_(1B) subunitwere published by Williams et al in 1992 (Science 257:389-395). Giventhe existence of several additional rat isoforms in a highly conservedgene family, it is surprising that additional human isoforms of theN-type calcium channel α_(1B) subunit have not been discovered. Suchisoforms would be useful for developing isoform-specific therapeutics.

SUMMARY OF THE INVENTION

[0006] The invention provides isolated nucleic acid molecules, uniquefragments of those molecules, expression vectors containing theforegoing, and host cells transfected with those molecules. Theinvention also provides isolated polypeptides and inhibitors of theforegoing nucleic acids and polypeptides which reduce voltage-gatedcalcium influx. The foregoing can be used in the diagnosis or treatmentof conditions characterized by increased or decreased human N-typecalcium channel hα_(1B+SFVG) subunit activity and can be used in methodsin which it is therapeutically useful to increase or decrease humanN-type calcium channel hα_(1B+SFVG) subunit activity such as treatmentsfor stroke, pain (e.g., neuropathic pain), traumatic brain injury andconditions characterized by increased or decreased voltage regulatedcalcium influx. Here, we present the identification of a novel humanN-type calcium channel α_(1B) subunit, hα_(1B+SFVG), which plays a rolein voltage-gated calcium influx.

[0007] It was discovered that a brain α_(1B) calcium channel subunitisoform (splice variant) contains a four amino acid insert relative topublished human α_(1B) calcium channel isoforms (SEQ ID NO: 5 [GenBankaccession number M94172], SEQ ID NO: 7 [GenBank accession numberM94173]). Surprisingly, this insert, SFVG (SEQ ID NO: 2, encoded by SEQID NO: 1), is similar but not identical to an insert found in a ratα_(1B) channel (GenBank accession number M92905). A significantproportion of the human N-type calcium channel α_(1B) subunit mRNA inbrain was found to be the hα_(1B+SFVG) sub-type; given the abundance ofits expression the isolation of this sub-type so long after theidentification of other α_(1B) isoforms is unexpected. TheSFVG-containing human N-type calcium channel hα_(1B+SFVG) subunit alsolacks an amino acid sequence, ET, which is present in published humanN-type calcium channel hα_(1B+SFVG) subunit isoforms (amino acids1557-1558 of SEQ ID NOs: 5 and 7).

[0008] The invention involves in one aspect an isolated human N-typecalcium channel α_(1B) subunit polypeptide which includes the amino acidsequence of SEQ ID NO: 2 (an hα_(1B+SFVG) polypeptide). In oneembodiment, the polypeptide comprises the amino acid sequence of SEQ IDNO: 4, and preferably consists of the amino acid sequence of SEQ ID NO:4. In another embodiment the hα_(1B+SFVG) calcium channel polypeptide isa fragment or variant of the foregoing polypeptides, wherein thefragment or variant includes the amino acid sequence of SEQ ID NO: 2 oradditions, deletions or substitutions thereof which confer the samefunction as SEQ ID NO: 2. Preferred variants include those havingadditions, substitutions or deletions relative to the human N-typecalcium channel hα_(1B+SFVG) SFVG subunit polypeptide sequence disclosedherein, particularly those variants which retain one or more of theactivities of the human N-type calcium channel hα_(1B+SFVG) subunit,including subunits with or without the ET exon sequence.

[0009] According to another aspect of the invention, an isolated nucleicacid molecule which encodes any of the foregoing human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide is provided. In certainembodiments, the nucleic acid molecule includes SEQ ID NO: 1. In onepreferred embodiment, the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides is encoded by a nucleic acid molecule whichcomprises the nucleotide sequence of SEQ ID NO: 3 (Williams et al.sequence +SFVG, −ET), and which preferably consists of the nucleotidesequence of SEQ ID NO: 3. In another embodiment the nucleic acid is anallele of the nucleic acid sequence of SEQ ID NO: 3.

[0010] In another aspect the invention is an expression vectorcomprising the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid molecule operably linked to a promoter. Also included within theinvention is a host cell transformed or transfected with the expressionvector.

[0011] According to another aspect of the invention, an agent whichselectively binds the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide or a nucleic acid that encodes the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide is provided. By “selectivelybinds” it is meant that the agent binds the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide or nucleic acid, or any fragmentthereof which retains the amino acids of SEQ ID NO: 2 or the nucleotidesof SEQ ID NO: 1, to a greater extent than the agent binds other humanN-type calcium channel α_(1B) subunit isoforms, and preferably does notbind other human N-type calcium channel α_(1B) subunit isoforms. In oneembodiment, the agent is a polypeptide which binds selectively to thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide. Thepolypeptide can be a monoclonal antibody, a polyclonal antibody, or anantibody fragment selected from the group consisting of a Fab fragment,a F(ab)₂ fragment and a fragment including a CDR3 region. In anotherembodiment, the agent is an antisense nucleic acid which selectivelybinds to a nucleic acid encoding the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. Preferably the foregoing agents areinhibitors (antagonists) or agonists of the calcium channel activity ofthe human N-type calcium channel hα_(1B+SFVG) subunit polypeptide.

[0012] According to another aspect of the inventions, a dominantnegative human N-type calcium channel hα_(1B+SFVG) subunit polypeptideis provided. The dominant negative polypeptide is an inhibitor of thefunction of the calcium channel.

[0013] The invention also provides compositions including any of theforegoing polypeptides, nucleic acids or agents in combination with apharmaceutically acceptable carrier.

[0014] In another aspect of the invention a method for inhibiting humanN-type calcium channel hα_(1B+SFVG) subunit activity in a mammalian cellis provided. The method involves the step of contacting the mammaliancell with an amount of a human N-type calcium channel hα_(1B+SFVG)subunit inhibitor effective to inhibit calcium influx in the mammaliancell. Preferably the inhibitor is selected from the group consisting ofa peptide or an antibody which selectively binds the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, an antisense nucleicacid which binds a nucleic acid encoding human N-type calcium channelhα_(1B+SFVG) subunit polypeptide and a dominant negative human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide.

[0015] According to still another aspect the invention, a method fortreating a subject having a stroke, pain (e.g., neuropathic pain), ortraumatic brain injury is provided. The method involves the step ofadministering to a subject in need of such treatment an inhibitor of thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide in anamount effective to inhibit voltage regulated calcium influx. In anotherembodiment of the foregoing methods, the inhibitor is administeredprophylactically to a subject at risk of having a stroke.

[0016] The human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides and nucleic acids which encode such polypeptides are usefulfor increasing the amount of human N-type calcium channel hα_(1B+SFVG)subunit polypeptides in a cell. Increasing the amount of human N-typecalcium channel hα_(1B+SFVG) subunit polypeptides in a cell results inincreased voltage regulated calcium influx. This is useful where it isdesired to increase the amount of voltage regulated calcium influx whichis mediated by a human N-type calcium channel.

[0017] Thus according to another aspect of the invention, a method forincreasing human N-type calcium channel hα_(1B+SFVG) subunit expressionin a cell is provided. The method involves the step of contacting thecell with a molecule selected from the group consisting of a humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid and a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide in an amounteffective to increase voltage regulated calcium influx in the cell. Incertain embodiments, the cell is contacted with one or more human N-typecalcium channel non-hα_(1B+SFVG) subunits, such as a β subunit, ornucleic acids encoding such hα_(1B+SFVG) subunits.

[0018] According to another aspect of the invention, a method forincreasing calcium channel voltage regulated calcium influx in a subjectis provided. The method involves the step of administering to a subjectin need of such treatment a molecule selected from the group consistingof a human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid anda human N-type calcium channel hα_(1B+SFVG) subunit polypeptide in anamount effective to increase voltage regulated calcium influx in thesubject.

[0019] According to a further aspect of the invention, a method foridentifying lead compounds for a pharmacological agent useful in thetreatment of disease associated with increased or decreased voltageregulated calcium influx mediated by a human N-type calcium channel isprovided. A cell or other membrane-encapsulated space comprising a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide is provided. Thecell or other membrane-encapsulated space preferably is loaded with acalcium-sensitive compound which is detectable in the presence ofcalcium. The cell or other membrane-encapsulated space is contacted witha candidate pharmacological agent under conditions which, in the absenceof the candidate pharmacological agent, cause a first amount of voltageregulated calcium influx into the cell or other membrane-encapsulatedspace. A test amount of voltage regulated calcium influx then isdetermined. For example, in a preferred embodiment, fluorescence of acalcium-sensitive compound then is detected as a measure of the voltageregulated calcium influx. If the test amount of voltage regulatedcalcium influx is less than the first amount, then the candidatepharmacological agent is a lead compound for a pharmacological agentwhich reduces voltage regulated calcium influx. If the test amount ofvoltage regulated calcium influx is greater than the first amount, thenthe candidate pharmacological agent is a lead compound for apharmacological agent which increases voltage regulated calcium influx.

[0020] In another aspect of the invention, methods for identifyingcompounds which selectively or preferentially bind a human N-typecalcium channel hα_(1B+SFVG) subunit isoform are provided. In oneembodiment, the method includes providing a first cell or membraneencapsulated space which expresses a human N-type calcium channelhα_(1B+SFVG) subunit isoform, and providing a second cell or membraneencapsulated space which expresses a human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform, wherein the second cell or membraneencapsulated space is identical to the first cell except for the α_(1B)isoform expressed. The first cell or membrane encapsulated space and thesecond cell or membrane encapsulated space are contacted with acompound, and the binding of the compound to the first cell or membraneencapsulated space and the second cell or membrane encapsulated space isdetermined. A compound which binds the first cell or membraneencapsulated space but does not bind the second cell or membraneencapsulated space is a compound which selectively binds the humanN-type calcium channel hα_(1B+SFVG) subunit isoform. A compound whichbinds the first cell or membrane encapsulated space in an amount greaterthan the compound binds the second cell or membrane encapsulated spaceis a compound which preferentially binds the human N-type calciumchannel hα_(1B+SFVG) subunit isoform. In another embodiment of themethod, a human N-type calcium channel hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid and a human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid areprovided and contacted with a compound. The binding of the compound tothe human N-type calcium channel hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid and the human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid the n isdetermined. A compound which binds the human N-type calcium channelhα_(1B+SFVG) subunit isoform polypeptide or nucleic acid but does notbind the human N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid is a compound which selectively binds thehuman N-type calcium channel hα_(1B+SFVG) subunit isoform polypeptide ornucleic acid. A compound which binds the human N-type calcium channelhα_(1B) +SFVG subunit isoform polypeptide or nucleic acid in an amountgreater than the human N-type calcium channel non-hα_(1B+SFVG) subunitisoform polypeptide or nucleic acid is a compound which preferentiallybinds the human N-type calcium channel hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid. Also included in the invention arecompounds identified using the foregoing methods.

[0021] According to another aspect of the invention, a method forselectively treating a subject having a condition characterized byaberrant brain neuronal calcium current is provided. The method includesthe step of administering to a subject in need of such treatment apharmacological agent which is selective for a human N-type calciumchannel hα_(1B+SFVG) subunit, in an amount effective to normalize theaberrant neuronal calcium current. Aberrant means a level of calciumcurrent (calcium influx) which is outside of a normal range asunderstood in the medical arts. Normalize means that the calcium currentis brought within the normal range.

[0022] Also presented herein is an identification of characteristics ofcertain calcium channel subunit isoforms with respect tovoltage-dependent activation. It has been discovered, surprisingly, thatthe presence or absence of an exon comprising the amino acids ET isimportant for the kinetics of channel activation. Thus, in still otheraspects of the invention, a variety of novel assays, screens,recombinant products, model systems (such as animal models) and methodsare provided which utilize the unexpected different activation functionsbetween and among the calcium channel subunit isoforms for theidentification of novel agents, treatments, etc. useful in themodulation of conditions which arise from or manifest differences inaction potential neurotransmitter release, voltage-dependent calciumchannel activation, and so on. For example, methods for theidentification of agents which alter activation potential dependentneurotransmitter release are provided. The methods include selecting anagent which binds a calcium channel isoform having or lacking a IVS3-S4ET exon as described herein, and determining calcium channel activationor activation potential dependent neurotransmitter release in thepresence and the absence of the agent. In some embodiments, candidatecompounds may be screened by such methods. The methods also can includemeasurement of these parameters in other calcium channel subunits whichmanifest such differences in activation kinetics, including subunits inwhich an NP exon is added or is substituted for the ET exon.

[0023] Use of the foregoing compositions in the preparation of amedicament, and particularly in the preparation of a medicament for thetreatment of stroke, pain (e.g., neuropathic pain), traumatic braininjury, or a condition which results from excessive or insufficientvoltage regulated calcium influx, is provided.

[0024] These and other aspects of the invention are described in greaterdetail below.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 shows that the presence of ET in domain IVS3-S4 of α_(1B)slows the rate of N-type Ca channel activation.

[0026]FIG. 2 shows the impact of alternative splicing in the S3-S4linkers of the α_(1B) subunit on action potential-dependent Ca influx ina model neuron.

[0027]FIG. 3 shows the results of a functional analysis of site-directedmutagenesis of ET splice site in domain IVS3-S4 of the α_(1B) subunit.

BRIEF DESCRIPTION OF THE SEQUENCES

[0028] SEQ ID NO: 1 is the nucleotide sequence of the human N-typecalcium channel hα_(1B+SFVG) subunit cDNA IIIS3-S4 “SFVG” site.

[0029] SEQ ID NO: 2 is the amino acid sequence of the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide IIIS3-S4 “SFVG” site.

[0030] SEQ ID NO: 3 is the nucleotide sequence of the human N-typecalcium channel hα_(1B+SFVG) subunit cDNA.

[0031] SEQ ID NO: 4 is the amino acid sequence of the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide.

[0032] SEQ ID NO: 5 is the nucleotide sequence of the coding region of ahuman α_(1B) calcium channel which lacks the IIIS3-S4 “SFVG” site (Priorart, GenBank accession number M94172).

[0033] SEQ ID NO: 6 is the amino acid sequence of a human α_(1B) calciumchannel which lacks the IIIS3-S4 “SFVG” site (Prior art, GenBankaccession number M94172).

[0034] SEQ ID NO: 7 is the nucleotide sequence of the coding region of ahuman α_(1B) calcium channel which lacks the IIIS3-S4 “SFVG” site (Priorart, GenBank accession number M94173).

[0035] SEQ ID NO: 8 is the amino acid sequence of a human α_(1B) calciumchannel which lacks the IIIS3-S4 “SFVG” site (Prior art, GenBankaccession number M94173).

[0036] SEQ ID NO: 9 is the nucleotide sequence of the coding region of arat α_(1B) calcium channel which contains a SFMG site (Prior art,GenBank accession number M92905).

[0037] SEQ ID NO: 10 is the amino acid sequence of a rat α_(1B) calciumchannel which contains a SFMG site (Prior art, GenBank accession numberM92905).

[0038] SEQ ID NO: 11 is the amino acid sequence of an ω-conotoxinpeptide from C. geographus.

[0039] SEQ ID NO: 12 is the amino acid sequence of ω-conotoxin peptidefrom C. magus.

[0040] SEQ ID NOS: 13-28 are primers for PCR and/or sequencing.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention in one aspect involves the identificationof a cDNA encoding a novel human isoform of the N-type calcium channel,referred to herein as the human N-type calcium channel hα_(1B+SFVG)subunit. As used herein, hα_(1B+SFVG) refers to any human N-type calciumchannel α_(1B) subunit clone that contains the SFVG sequence set forthin SEQ ID NO: 2. The nucleotide sequence of the human N-type calciumchannel hα_(1B+SFVG) subunit insert, the IIIS3-S4 “SFVG” site, ispresented as SEQ ID NO: 1, and the amino acid sequence of the humanN-type calcium channel hα_(1B+SFVG) subunit insert, the IIIS3-S4 “SFVG”site, is presented as SEQ ID NO: 2. The 12 nucleotides of SEQ ID NO: 1are inserted immediately following nt 3855 in the coding sequence of thehuman N-type calcium channel α_(1B) subunit in the codon encoding aminoacid 1237, such that the four amino acids of SEQ ID NO: 2 are insertedin the polypeptide after amino acid 1237 (see SEQ ID NO: 3). The closelyrelated human N-type calcium channel α_(1B) subunit, which does notcontain the IIIS3-S4 “SFVG” site, was deposited in GenBank underaccession numbers M94172 and M94173 (SEQ ID NOs: 5-8). A related ratN-type calcium channel α_(1B-b) subunit was deposited in GenBank underaccession number M92905 (SEQ ID NOs: 9 and 10). Surprisingly, the aminoacid sequence of the human N-type calcium channel hα_(1B+SFVG) subunitdiffers from the rat amino acid sequence in the SFVG site, whichsequence is located in an area of the molecule in which the human andrat amino acid sequences are otherwise 100% identical. This speciesdifference in the very highly conserved protein domain of the humanN-type calcium channel hα_(1B+SFVG) subunit r entirely unexpected, andpermits the screening of compounds which selectively bind to and/ormodulate the human N-type calcium channel hα_(1B+SFVG) subunit. Becausethe present human N-type calcium channel hα_(1B+SFVG) subunit is asplice variant of other human N-type calcium channel α_(1B) subunits, itis apparent that the invention is meant to embrace human N-type calciumchannel α_(1B) subunit variants which vary by alternative splicing ofsequences other than the SFVG (SEQ ID NO: 2) insert. For example, theinvention embraces polypeptides which contain or do not contain an Alaresidue immediately following amino acid position 414 of SEQ ID NO: 3,or a Glu-Thr insert (ET in single letter code) at amino acid positions1557-1558 (see, e.g., SEQ ID NO: 6), as well as nucleic acid moleculesencoding such splice variant polypeptides. As shown in the Examples, thehα_(1B+SFVG) subunit is a significant portion of the α_(1B) calciumchannel expressed in human brain, and is differentially distributed indifferent parts of the brain. This opens the possibility for theselective treatment of disorders which involve those parts of the brain.

[0042] The invention involves in one aspect human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids and polypeptides, as well astherapeutics relating thereto. The invention also embraces isolatedfunctionally equivalent variants, useful analogs and fragments of theforegoing nucleic acids and polypeptides; complements of the foregoingnucleic acids; and molecules which selectively bind the foregoingnucleic acids and polypeptides.

[0043] The human N-type calcium channel hα_(1B+SFVG) subunit nucleicacids and polypeptides of the invention are isolated. The term“isolated”, as used herein in reference to a nucleic acid molecule,means a nucleic acid sequence: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) synthesized by, for example,chemical synthesis; (iii) recombinantly produced by cloning; or (iv)purified, as by cleavage and electrophoretic or chromatographicseparation. The term “isolated”, as used herein in reference to apolypeptide, means a polypeptide encoded by an isolated nucleic acidsequence, as well as polypeptides synthesized by, for example, chemicalsynthetic methods, and polypeptides separated from biological materials,and then purified, using conventional protein analytical or preparatoryprocedures, to an extent that permits them to be used according to themethods described herein.

[0044] As used herein a human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid refers to an isolated nucleic acid molecule whichcodes for a human N-type calcium channel hα_(1B+SFVG) subunit. HumanN-type calcium channel hα_(1B+SFVG) subunit nucleic acids are thosenucleic acid molecules which code for human N-type calcium channelhα_(1B+SFVG) subunit polypeptides which include the sequence of SEQ IDNO: 2. The nucleic acid molecules include the nucleotide sequence of SEQID NO: 1 and nucleotide sequences which differ from the sequence of SEQID NO: 1 in codon sequence due to the degeneracy of the genetic code.The human N-type calcium channel hα_(1B+SFVG) subunit nucleic acids ofthe invention also include alleles of the foregoing nucleic acids, aswell as fragments of the foregoing nucleic acids, provided that theallele or fragment encodes the amino acid sequence of SEQ ID NO: 2. Suchfragments can be used, for example, as probes in hybridization assaysand as primers in a polymerase chain reaction (PCR). Preferred humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acids include thenucleic acid sequence of SEQ ID NO: 1. Complements of the foregoingnucleic acids also are embraced by the invention.

[0045] As used herein “human N-type calcium channel hα_(1B+SFVG) subunitactivity” refers to an ability of a molecule to modulate voltageregulated calcium influx. A molecule which inhibits human N-type calciumchannel hα_(1B+SFVG) subunit activity (an antagonist) is one whichinhibits voltage regulated calcium influx via this calcium channel and amolecule which increases human N-type calcium channel hα_(1B+SFVG)subunit activity (an agonist) is one which increases voltage regulatedcalcium influx via this calcium channel. Changes in human N-type calciumchannel hα_(1B+SFVG) subunit activity can be measured by changes involtage regulated calcium influx by in vitro assays such as thosedisclosed herein, including patch-clamp assays and assays employingcalcium sensitive fluorescent compounds such as fura-2.

[0046] Alleles of the human N-type calcium channel hα_(1B+SFVG) subunitnucleic acids of the invention can be identified by conventionaltechniques. For example, alleles of human N-type calcium channelhα_(1B+SFVG) subunit can be isolated by hybridizing a probe whichincludes SEQ ID NO: 1 under stringent conditions with a cDNA library andselecting positive clones. Thus, an aspect of the invention is thosenucleic acid sequences which code for human N-type calcium channelhα_(1B+SFVG) subunit polypeptides and which hybridize to a nucleic acidmolecule consisting of SEQ ID NO: 1 under stringent conditions. The term“stringent conditions” as used herein refers to parameters with whichthe art is familiar. Nucleic acid hybridization parameters may be foundin references which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15 M sodium chloride/0.015 M sodium citrate, pH7;SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraceticacid. After hybridization, the membrane upon which the DNA istransferred is washed at 2×SSC at room temperature and then at0.1×SSC/0.1×SDS at temperatures up to 65° C.

[0047] There are other conditions, reagents, and so forth which can beused, which result in a similar degree of stringency. The skilledartisan will be familiar with such conditions, and thus they are notgiven here. It will be understood, however, that the skilled artisanwill be able to manipulate the conditions in a manner to permit theclear identification of alleles of human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids of the invention. The skilled artisanalso is familiar with the methodology for screening cells and librariesfor expression of such molecules which then are routinely isolated,followed by isolation of the pertinent nucleic acid molecule andsequencing.

[0048] In screening for human N-type calcium channel hα_(1B+SFVG)subunit nucleic acids, a Southern blot may be performed using theforegoing stringent conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film to detect the radioactivesignal.

[0049] The human N-type calcium channel hα_(1B+SFVG) subunit nucleicacids of the invention also include degenerate nucleic acids whichinclude alternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide. Similarly, nucleotidesequence triplets which encode other amino acid residues include, butare not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC,CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT(threonine codons); AAC and AAT (asparagine codons); and ATA, ATC andATT (isoleucine codons). Other amino acid residues may be encodedsimilarly by multiple nucleotide sequences. Thus, the invention embracesdegenerate nucleic acids that differ from the biologically isolatednucleic acids in codon sequence due to the degeneracy of the geneticcode.

[0050] The invention also provides isolated fragments of SEQ ID NO: 3which include the nucleotide sequence of SEQ ID NO: 1. The fragments canbe used as probes in Southern blot assays to identify such nucleicacids, or can be used in amplification assays such as those employingPCR. Smaller fragments are those comprising 12, 13, 14, 15, 16, 17, 18,20, 22, 25, 30, 40, 50, or 75 nucleotides, and every integertherebetween and are useful e.g. as primers for nucleic acidamplification procedures. As known to those skilled in the art, largerprobes such as 200, 250, 300, 400 or more nucleotides are preferred forcertain uses such as Southern blots, while smaller fragments will bepreferred for uses such as PCR. Fragments also can be used to producefusion proteins for generating antibodies or determining binding of thepolypeptide fragments. Likewise, fragments can be employed to producenon-fused fragments of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides, useful, for example, in the preparation ofantibodies, in immunoassays, and the like. The foregoing nucleic acidfragments further can be used as antisense molecules to inhibit theexpression of human N-type calcium channel hα_(1B+SFVG) subunit nucleicacids and polypeptides, particularly for therapeutic purposes asdescribed in greater detail below.

[0051] The invention also includes functionally equivalent variants ofthe human N-type calcium channel hα_(1B+SFVG) subunit, which includevariant nucleic acids and polypeptide which retain one or more of thefunctional properties of the human N-type calcium channel hα_(1B+SFVG)subunit, but always including SEQ ID NO: 2. For example, variantsinclude a fusion protein which includes the extracellular andtransmembrane domains of the human N-type calcium channel hα_(1B+SFVG)subunit (including SEQ ID NO: 2), which retains the ability to bindligand and/or transduce a voltage gated calcium current. Still otherfunctionally equivalent variants include variants of SEQ ID NO: 2 whichretain functions of subunit including SEQ ID NO: 2. Functionallyequivalent variants also include a human N-type calcium channelhα_(1B+SFVG) subunit which has had a portion of the extracellular domain(but not SEQ ID NO: 2) removed or replaced by a similar domain fromanother calcium channel α₁ subunit (e.g. a “domain-swapping” variant).Other functionally equivalent variants will be known to one of ordinaryskill in the art, as will methods for preparing such variants. Theactivity of a functionally equivalent variant can be determined usingthe methods provided herein, in Lin et al., Neuron 18:153-166, 1997, andin U.S. Pat. No. 5,429,921. Such variants are useful, inter alia, inassays for identification of compounds which bind and/or regulate thecalcium influx function of the human N-type calcium channel hα_(1B+SFVG)subunit, and for determining the portions of the human N-type calciumchannel hα_(1B+SFVG) subunit which are required for calcium influxactivity.

[0052] Variants which are non-functional also can be prepared asdescribed above. Such variants are useful, for example, as negativecontrols in experiments testing subunit activity, and as inhibition ofN-type calcium channel activity.

[0053] A human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid,in one embodiment, is operably linked to a gene expression sequencewhich directs the expression of the human N-type calcium channelhα_(1B+SFVG) subunit nucleic acid within a eukaryotic or prokaryoticcell. The “gene expression sequence” is any regulatory nucleotidesequence, such as a promoter sequence or promoter-enhancer combination,which facilitates the efficient transcription and translation of thehuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid to whichit is operably linked. The gene expression sequence may, for example, bea mammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus, papilloma virus, adenovirus, humanimmunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, thelong terminal repeats (LTR) of Moloney murine leukemia virus and otherretroviruses, and the thymidine kinase promoter of herpes simplex virus.Other constitutive promoters are known to those of ordinary skill in theart. The promoters useful as gene expression sequences of the inventionalso include inducible promoters. Inducible promoters are expressed inthe presence of an inducing agent. For example, the metallothioneinpromoter is induced to promote transcription and translation in thepresence of certain metal ions. Other inducible promoters are known tothose of ordinary skill in the art.

[0054] In general, the gene expression sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid. The gene expression sequences optionally includes enhancersequences or upstream activator sequences as desired.

[0055] The human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid sequence and the gene expression sequence are said to be “operablylinked” when they are covalently linked in such a way as to place thetranscription and/or translation of the human N-type calcium channelhα_(1B+SFVG) subunit coding sequence under the influence or control ofthe gene expression sequence. If it is desired that the human N-typecalcium channel hα_(1B+SFVG) subunit sequence be translated into afunctional protein, two DNA sequences are said to be operably linked ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the human N-type calcium channel hα_(1B+SFVG)subunit sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the human N-type calcium channelhα_(1B+SFVG) subunit sequence, or (3) interfere with the ability of thecorresponding RNA transcript to be translated into a protein. Thus, agene expression sequence would be operably linked to a human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid sequence if the geneexpression sequence were capable of effecting transcription of thathuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid sequencesuch that the resulting transcript might be translated into the desiredprotein or polypeptide.

[0056] The human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid and the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide (including the human N-type calcium channel hα_(1B+SFVG)subunit inhibitors described below) of the invention can be delivered tothe eukaryotic or prokaryotic cell alone or in association with avector. In its broadest sense, a “vector” is any vehicle capable offacilitating: (1) delivery of a human N-type calcium channelhα_(1B+SFVG) subunit nucleic acid or polypeptide to a target cell or (2)uptake of a human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid or polypeptide by a target cell. Preferably, the vectors transportthe human N-type calcium channel hα_(1B+SFVG) subunit nucleic acid orpolypeptide into the target cell with reduced degradation relative tothe extent of degradation that would result in the absence of thevector. Optionally, a “targeting ligand” can be attached to the vectorto selectively deliver the vector to a cell which expresses on itssurface the cognate receptor (e.g. a receptor, an antigen recognized byan antibody) for the targeting ligand. In this manner, the vector(containing a human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid or a human N-type calcium channel hα_(1B+SFVG) subunit polypeptide)can be selectively delivered to a specific cell. In general, the vectorsuseful in the invention are divided into two classes: biological vectorsand chemical/physical vectors. Biological vectors are more useful fordelivery/uptake of human N-type calcium channel hα_(1B+SFVG) subunitnucleic acids to/by a target cell. Chemical/physical vectors are moreuseful for delivery/uptake of human N-type calcium channel hα_(1B+SFVG)subunit nucleic acids or human N-type calcium channel hα_(1B+SFVG)subunit proteins to/by a target cell.

[0057] Biological vectors include, but are not limited to, plasmids,phagemids, viruses, other vehicles derived from viral or bacterialsources that have been manipulated by the insertion or incorporation ofthe nucleic acid sequences of the invention, and free nucleic acidfragments which can be attached to the nucleic acid sequences of theinvention. Viral vectors are a preferred type of biological vector andinclude, but are not limited to, nucleic acid sequences from thefollowing viruses: retroviruses, such as Moloney murine leukemia virus;Harvey murine sarcoma virus; murine mammary tumor virus; Rous sarcomavirus; adenovirus; adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vacciniavirus; and polio virus. One can readily employ other vectors not namedbut known in the art.

[0058] Preferred viral vectors are based on non-cytopathic eukaryoticviruses in which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. In general, theretroviruses are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., “Gene Transfer and Expression, A Laboratory Manual, ” W.H. Freeman C. O., New York (1990) and Murry, E. J. Ed. “Methods inMolecular Biology,” vol. 7, Humana Press, Inc., Clifton, N.J. (1991).

[0059] Another preferred virus for certain applications is theadeno-associated virus, a double-stranded DNA virus. Theadeno-associated virus can be engineered to be replication-deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages, such as heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

[0060] Expression vectors containing all the necessary elements forexpression are commercially available and known to those skilled in theart. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide or fragment or variant thereof. Thatheterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

[0061] Preferred systems for mRNA expression in mammalian cells arethose such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.) thatcontain a selectable marker such as a gene that confers G418 resistance(which facilitates the selection of stably transfected cell lines) andthe human cytomegalovirus (CMV) enhancer-promoter sequences.Additionally, suitable for expression in primate or canine cell lines isthe pCEP4 vector (Invitrogen), which contains an Epstein Barr virus(EBV) origin of replication, facilitating the maintenance of plasmid asa multicopy extrachromosomal element. Another expression vector is thepEF-BOS plasmid containing the promoter of polypeptide Elongation Factor1α, which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992).

[0062] In addition to the biological vectors, chemical/physical vectorsmay be used to deliver a human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid or polypeptide to a target cell and facilitateuptake thereby. As used herein, a “chemical/physical vector” refers to anatural or synthetic molecule, other than those derived frombacteriological or viral sources, capable of delivering the isolatedhuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid orpolypeptide to a cell.

[0063] A preferred chemical/physical vector of the invention is acolloidal dispersion system. Colloidal dispersion systems includelipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. A preferred colloidal system of the inventionis a liposome. Liposomes are artificial membrane vesicles which areuseful as a delivery vector in vivo or in vitro. It has been shown thatlarge unilamellar vesicles (LUV), which range in size from 0.2-4.0 μ canencapsulate large macromolecules. RNA, DNA, and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, et al., Trends Biochem. Sci., v. 6, p.77 (1981)). In order for a liposome to be an efficient nucleic acidtransfer vector, one or more of the following characteristics should bepresent: (1) encapsulation of the nucleic acid of interest at highefficiency with retention of biological activity; (2) preferential andsubstantial binding to a target cell in comparison to non-target cells;(3) delivery of the aqueous contents of the vesicle to the target cellcytoplasm at high efficiency; and (4) accurate and effective expressionof genetic information.

[0064] Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Ligands which may be useful for targeting aliposome to a particular cell will depend on the particular cell ortissue type. Additionally when the vector encapsulates a nucleic acid,the vector may be coupled to a nuclear targeting peptide, which willdirect the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid to the nucleus of the host cell.

[0065] Liposomes are commercially available from Gibco BRL, for example,as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipidssuch as N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, V. 3, p. 235-241 (1985).

[0066] Other exemplary compositions that can be used to facilitateuptake by a target cell of the human N-type calcium channel hα_(1B+SFVG)subunit nucleic acids include calcium phosphate and other chemicalmediators of intracellular transport, microinjection compositions,electroporation and homologous recombination compositions (e.g., forintegrating a human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid into a preselected location within a target cell chromosome).

[0067] The invention also embraces so-called expression kits, whichallow the artisan to prepare a desired expression vector or vectors.Such expression kits include at least separate portions of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

[0068] It will also be recognized that the invention embraces the use ofthe human N-type calcium channel hα_(1B+SFVG) subunit cDNA sequences inexpression vectors, as well as to transfect host cells and cell lines,be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., COS cells,yeast expression systems and recombinant baculovirus expression ininsect cells). Especially useful are mammalian cells such as human, pig,goat, primate, etc. They may be of a wide variety of tissue types, andinclude primary cells and cell lines. Specific examples include neuronalcells including PC 12 cells, Xenopus oocytes, bone marrow stem cells andembryonic stem cells. The expression vectors require that the pertinentsequence, i.e., those nucleic acids described supra, be operably linkedto a promoter.

[0069] The invention also provides isolated human N-type calcium channelhα_(1B+SFVG) subunit polypeptides which include the amino acid sequenceof SEQ ID NO: 2, encoded by the human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids described above. The preferred humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide has the aminoacid sequence of SEQ ID NO: 4. Human N-type calcium channel hα_(1B+SFVG)subunit polypeptides also embrace alleles, functionally equivalentvariants and analogs (those non-allelic polypeptides which vary in aminoacid sequence from SEQ ID NO: 4 by 1, 2, 3, 4, 5, or more amino acids)provided that such polypeptides include the amino acids of SEQ ID NO: 2and retain human N-type calcium channel hα_(1B+SFVG) subunit activity,and fragments of SEQ ID NO: 4 which include SEQ ID NO: 2. Non-functionalvariants also are embraced by the invention; these are useful asantagonists of calcium channel function, as negative controls in assays,and the like. Such alleles, variants, analogs and fragments are useful,for example, alone or as fusion proteins for a variety of purposes suchas to generate antibodies, or as a component of an immunoassay.

[0070] Fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide, in particular voltageregulated calcium influx. Other functional capabilities which can beretained in a fragment of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide include interaction with antibodies and interactionwith other polypeptides (such as other subunits of the human N-typecalcium channel). Those skilled in the art are well versed in methodsfor selecting fragments which retain a functional capability of thehuman N-type calcium channel hα_(1B+SFVG) subunit. Confirmation of thefunctional capability of the fragment can be carried out by synthesis ofthe fragment and testing of the capability according to standardmethods. For example, to test the voltage regulated calcium influx of ahuman N-type calcium channel hα_(1B+SFVG) subunit fragment, one insertsor expresses the fragment in a cell in which calcium influx can bemeasured. Such methods, which are standard in the art, are describedfurther in the examples.

[0071] The invention embraces variants of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides described above. As usedherein, a “variant” of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide is a polypeptide which contains one or moremodifications to the primary amino acid sequence of a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide. Modifications whichcreate a human N-type calcium channel hα_(1B+SFVG) subunit variant canbe made to a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide for a variety of reasons, including 1) to reduce oreliminate an activity of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide, such as voltage gated calcium influx; 2) to enhancea property of a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, such as protein stability in an expression system or thestability of protein-protein binding; 3) to provide a novel activity orproperty to a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, such as addition of an antigenic epitope or addition of adetectable moiety; or 4) to establish that an amino acid substitutiondoes or does not affect voltage gated calcium influx. Modifications to ahuman hα_(1B+SFVG) calcium channel polypeptide are typically made to thenucleic acid which encodes the human N-type calcium channel hα_(1B+SFVG)subunit polypeptide, and can include deletions, point mutations,truncations, amino acid substitutions and additions of amino acids ornon-amino acid moieties. Alternatively, modifications can be madedirectly to the polypeptide, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as biotin, addition of afatty acid, and the like. Modifications also embrace fusion proteinscomprising all or part of the human N-type calcium channel hα_(1B+SFVG)subunit amino acid sequence, but always including SEQ ID NO: 2. One ofskill in the art will be familiar with methods for predicting the effecton protein conformation of a change in protein sequence, and can thus“design” a variant human N-type calcium channel hα_(1B+SFVG) subunitaccording to known methods. One example of such a method is described byDahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can bedesigned de novo. The method can be applied to a known protein to vary aonly a portion of the polypeptide sequence. By applying thecomputational methods of Dahiyat and Mayo, specific variants of a cancerassociated antigen polypeptide can be proposed and tested to determinewhether the variant retains a desired conformation.

[0072] Variants include human N-type calcium channel hα_(1B+SFVG)subunit polypeptides which are modified specifically to alter a featureof the polypeptide unrelated to its physiological activity. For example,cysteine residues can be substituted or deleted to prevent unwanteddisulfide linkages. Similarly, certain amino acids can be changed toenhance expression of a human N-type calcium channel subunit polypeptideby eliminating proteolysis by proteases in an expression system (e.g.,dibasic amino acid residues in yeast expression systems in which KEX2protease activity is present).

[0073] Mutations of a nucleic acid which encode a human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide preferably preserve the aminoacid reading frame of the coding sequence, and preferably do not createregions in the nucleic acid which are likely to hybridize to formsecondary structures, such as hairpins or loops, which can bedeleterious to expression of the variant polypeptide.

[0074] Mutations can be made by selecting an amino acid substitution, orby random mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with a desired property. Further mutations can be made tovariants (or to non-variant human N-type calcium channel hα_(1B+SFVG)subunit polypeptides) which are silent as to the amino acid sequence ofthe polypeptide, but which provide preferred codons for translation in aparticular host. The preferred codons for translation of a nucleic acidin, e.g., E. coli, are well known to those of ordinary skill in the art.Still other mutations can be made to the noncoding sequences of a humanN-type calcium channel hα_(1B+SFVG) subunit gene or EDNA clone toenhance expression of the polypeptide.

[0075] The activity of variants of human N-type calcium channelhα_(1B+SFVG) subunit polypeptides can be tested by cloning the geneencoding the variant human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide into a bacterial or mammalian expression vector, introducingthe vector into an appropriate host cell, expressing the variant humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide, and testing fora functional capability of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides as disclosed herein. For example, the variant humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide can be testedfor ability to provide voltage regulated calcium influx, as set forthbelow in the examples. Preparation of other variant polypeptides mayfavor testing of other activities, as will be known to one of ordinaryskill in the art.

[0076] The skilled artisan will also realize that conservative aminoacid substitutions may be made in human N-type calcium channelhα_(1B+SFVG) subunit polypeptides to provide functionally equivalentvariants of the foregoing polypeptides, i.e., variants which retain thefunctional capabilities of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides. As used herein, a “conservative amino acidsubstitution” refers to an amino acid substitution which does not alterthe relative charge or size characteristics of the polypeptide in whichthe amino acid substitution is made. Variants can be prepared accordingto methods for altering polypeptide sequence known to one of ordinaryskill in the art such as are found in references which compile suchmethods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds., Second Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplaryfunctionally equivalent variants of the human N-type calcium channelhα_(1B+SFVG) subunit polypeptides include conservative amino acidsubstitutions of SEQ ID NO: 4, but excluding the portion of thepolypeptide consisting of SEQ ID NO: 2 (SFVG). Conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0077] Conservative amino-acid substitutions in the amino acid sequenceof human N-type calcium channel hα_(1B+SFVG) subunit polypeptide toproduce functionally equivalent variants of human N-type calcium channelhα_(1B+SFVG) subunit polypeptides typically are made by alteration ofthe nucleic acid sequence encoding human N-type calcium channelhα_(1B+SFVG) subunit polypeptides (e.g., SEQ ID NO: 3). Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, such as a leucinezipper domain, the substitutions can be made by directly synthesizingthe peptide. The activity of functionally equivalent fragments of humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides can be testedby cloning the gene encoding the altered human N-type calcium channelhα_(1B+SFVG) subunit polypeptide into a bacterial or mammalianexpression vector, introducing the vector into an appropriate host cell,expressing the altered human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, and testing for the ability of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide to transduce voltage regulatedcalcium influx. Peptides which are chemically synthesized can be testeddirectly for function.

[0078] A variety of methodologies well-known to the skilled practitionercan be utilized to obtain isolated human N-type calcium channelhα_(1B+SFVG) subunit molecules. The polypeptide may be purified fromcells which naturally produce the polypeptide by chromatographic meansor immunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the polypeptide. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded polypeptide. Translationof mRNA in cell-free extracts such as the reticulocyte lysate systemalso may be used to produce polypeptide. Those skilled in the art alsocan readily follow known methods for isolating human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides. These include, but are notlimited to, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography and immune-affinity chromatography.

[0079] The invention as described herein has a number of uses, some ofwhich are described elsewhere herein. For example, the invention permitsisolation of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide molecules containing the amino acid sequence of SEQ ID NO: 2by e.g., expression of a recombinant nucleic acid to produce largequantities of polypeptide which may be isolated using standardprotocols. As another example, the isolation of the human N-type calciumchannel hα_(1B+SFVG) subunit gene makes it possible for the artisan todiagnose a disorder characterized by loss of expression of human N-typecalcium channel hα_(1B+SFVG) subunit. These methods involve determiningexpression of the human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid, and/or human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes.

[0080] The invention also embraces agents which bind selectively to thehuman N-type calcium channel hα_(1B+SFVG) subunit (having or encodingSEQ ID NO: 2) and agents which bind preferentially to the human N-typecalcium channel hα_(1B+SFVG) subunit (having or encoding SEQ ID NO: 2)as well as agents which bind to variants and fragments of thepolypeptides and nucleic acids as described herein. Selective bindingmeans that the agent binds to the human N-type calcium channelhα_(1B+SFVG) subunit but not to human N-type calcium channelnon-hα_(1B+SFVG) subunits (i.e., those subunits which do not have orencode SEQ ID NO: 2). Preferential binding means that the agent bindsmore to the human N-type calcium channel hα_(1B+SFVG) subunit than tohuman N-type calcium channel non-hα_(1B+SFVG) subunit, e.g., the agentbinds with greater affinity or avidity to the human N-type calciumchannel hα_(1B+SFVG) subunit having or encoding SEQ ID NO: 2. The agentsinclude polypeptides which bind to human N-type calcium channelhα_(1B+SFVG) subunit, and antisense nucleic acids, both of which aredescribed in greater detail below. The agents can inhibit or increasehuman N-type calcium channel hα_(1B+SFVG) subunit activity (antagonistsand agonists, respectively).

[0081] Some of the agents are inhibitors. A human N-type calcium channelhα_(1B+SFVG) subunit inhibitor is an agent that inhibits human N-typecalcium channel hα_(1B+SFVG) subunit mediated voltage gated calciuminflux. Human N-type calcium channel hα_(1B+SFVG) subunit inhibitorsalso include dominant negative peptides and known N-type calcium channelinhibitors including the ω-conotoxin peptides and derivative thereofsuch as ziconotide (SNX-111). Small organic molecule calcium channelinhibitors, such as fluspirilene, NNC09-0026(−)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-phenoxy)methyl] piperidinedihydrochloride); SB 201823-A(4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidinehydrochloride); NS649 (2-amino-i-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole);CNS 1237 (N-acenaphthyl-N′-4-methoxynaphth-1-yl guanidine) and riluzolemay also exhibit specificity for the human N-type calcium channelhα_(1B+SFVG) subunit.

[0082] Calcium influx assays can be performed to screen and/or determinewhether a human N-type calcium channel hα_(1B+SFVG) subunit inhibitorhas the ability to inhibit human N-type calcium channel hα_(1B+SFVG)subunit activity, and whether the inhibition is selective. As usedherein, “inhibit” refers to inhibiting by at least 10% voltage gatedcalcium influx, preferably inhibiting by at least 25% voltage gatedcalcium influx, and more preferably inhibiting by at least 40% voltagegated calcium influx as measured by any of the methods well known in theart. An exemplary assay of voltage gated calcium influx is describedbelow in the Examples.

[0083] Inhibitors may selectively inhibit hα_(1B+SFVG) based on thestate of depolarization of the membrane with which the hα_(1B+SFVG) isassociated. It is well known that certain compounds preferentially bindto voltage-gated calcium channels at particular voltages. For example,dihydropyridine compounds preferentially bind to L-type voltage-gatedcalcium channels when the membrane is depolarized. Bean (Proc. Nat'l.Acad. Sci. 81:6388, 1984) described the binding of nitrendipine tocardiac L-type channels only when the membrane is depolarized. Similarresults have been found for nimodipine action in sensory neurons(McCarthy & TanPiengco, J. Neurosci. 12:2225, 1992).

[0084] Activators of human N-type calcium channel hα_(1B+SFVG) activityalso are enhanced by the invention. Activators may be identified and/ortested using methods described above for inhibitors. The SFVG site islocated in a portion of the hα_(1B+SFVG) channel which is important forvoltage dependent gating of Ca²⁺ influx. Therefore, in screening formodulators of hα_(1B+SFVG) including inhibitors and activators (i.e.antagonists and agonists), it is preferred that compounds (e.g.libraries of potential channel inhibitors) are tested for modulation ofhα_(1B+SFVG) activity at a variety of voltages which cause partial orcomplete membrane depolarization, or hyperpolarization. These assays areconducted according to standard procedures of testing calcium channelfunction (e.g. patch clamping, fluorescent Ca²⁺ influx assays) whichrequire no more than routine experimentation. Using such methods,modulators of hα_(1B+SFVG) activity which are active at particularvoltages (e.g. complete membrane depolarization) can be identified. Suchcompounds are useful for selectively modulating calcium channel activityin conditions which may display voltage dependence. For example,following a stroke membranes are depolarized and such compounds may beactive in selectively blocking calcium channel activity for treatment ofstroke. Other uses will be apparent to one of ordinary skill in the art.

[0085] In one embodiment the human N-type calcium channel hα_(1B+SFVG)subunit inhibitor is an antisense oligonucleotide that selectively bindsto a human N-type calcium channel hα_(1B+SFVG) subunit nucleic acidmolecule, to reduce the expression of human N-type calcium channelhα_(1B+SFVG) subunit in a cell. This is desirable in virtually anymedical condition wherein a reduction of human N-type calcium channelhα_(1B+SFVG) subunit activity is desirable, e.g., voltage gated calciuminflux.

[0086] As used herein, the term “antisense oligonucleotide” or“antisense” describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO: 1, or upon allelic or homologousgenomic and/or cDNA sequences, one of skill in the art can easily chooseand synthesize any of a number of appropriate antisense molecules foruse in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably,the antisense oligonucleotides comprise a complementary sequence of20-30 bases. Although oligonucleotides may be chosen which are antisenseto any region of the gene or mRNA transcripts, in preferred embodimentsthe antisense oligonucleotides correspond to N-terminal or 5′ upstreamsites such as translation initiation, transcription initiation orpromoter sites. In addition, 3′-untranslated regions may be targeted.Targeting to mRNA splicing sites has also been used in the art but maybe less preferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol Neurobiol.14(5):439-457, 1994) and at which polypeptides are not expected to bind.Thus, the present invention also provides for antisense oligonucleotideswhich are complementary to allelic or homologous cDNAs and genomic DNAscorresponding to human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid containing SEQ ID NO: 1.

[0087] In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

[0088] In preferred embodiments, however, the antisense oligonucleotidesof the invention also may include “modified” oligonucleotides. That is,the oligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

[0089] The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

[0090] The term “modified oligonucleotide” also encompassesoligonucleotides with a covalently modified base and/or sugar. Forexample, modified oligonucleotides include oligonucleotides havingbackbone sugars which are covalently attached to low molecular weightorganic groups other than a hydroxyl group at the 3′ position and otherthan a phosphate group at the 5′ position. Thus modifiedoligonucleotides may include a 2′-O-alkylated ribose group. In addition,modified oligonucleotides may include sugars such as arabinose insteadof ribose. The present invention, thus, contemplates pharmaceuticalpreparations containing modified antisense molecules that arecomplementary to and hybridizable with, under physiological conditions,nucleic acids encoding human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides, together with pharmaceutically acceptable carriers.

[0091] Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardpharmaceutically acceptable carriers which are known in the art. Thecompositions should be sterile and contain a therapeutically effectiveamount of the antisense oligonucleotides in a unit of weight or volumesuitable for administration to a patient. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Thecharacteristics of the carrier will depend on the route ofadministration. Pharmaceutically acceptable carriers include diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswhich are well known in the art.

[0092] Agents which bind human N-type calcium channel hα_(1B+SFVG)subunit also include binding peptides which bind to the human N-typecalcium channel hα_(1B+SFVG) subunit and complexes containing the humanN-type calcium channel hα_(1B+SFVG) subunit. When the bindingpolypeptides are inhibitors, the polypeptides bind to and inhibit theactivity of human N-type calcium channel hα_(1B+SFVG) subunit. Todetermine whether a human N-type calcium channel hα_(1B+SFVG) subunitbinding peptide binds to human N-type calcium channel hα_(1B+SFVG)subunit any known binding assay may be employed. For example, thepeptide may be immobilized on a surface and then contacted with alabeled human N-type calcium channel hα_(1B+SFVG) subunit. The amount ofhuman N-type calcium channel hα_(1B+SFVG) subunit which interacts withthe human N-type calcium channel hα_(1B+SFVG) subunit binding peptide orthe amount which does not bind to the human N-type calcium channelhα_(1B+SFVG) subunit binding peptide may then be quantitated todetermine whether the human N-type calcium channel hα_(1B+SFVG) subunitbinding peptide binds to human N-type calcium channel hα_(1B+SFVG)subunit. Further, the binding of a human N-type calcium channelhα_(1B+SFVG) subunit and a human N-type calcium channel non-hα_(1B+SFVG)subunit can be compared to determine if the binding peptide bindsselectively or preferentially.

[0093] The human N-type calcium channel hα_(1B+SFVG) subunit bindingpeptides include peptides of numerous size and type that bindselectively or preferentially to human N-type calcium channelhα_(1B+SFVG) subunit polypeptides, and complexes of both human N-typecalcium channel hα_(1B+SFVG) subunit polypeptides and their bindingpartners. These peptides may be derived from a variety of sources. Forexample, binding peptides include known N-type calcium channelinhibitors such as the ω-conotoxin peptides GVIA SEQ ID NO: 11 (from C.geographus) and MVIIA SEQ ID NO: 12 (from C. magus). Other such humanN-type calcium channel hα_(1B+SFVG) subunit binding peptides can beprovided by modifying the foregoing peptides or by screening degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptoids and non-peptidesynthetic moieties.

[0094] Phage display can be particularly effective in identifyingbinding peptides useful according to the invention. Briefly, oneprepares a phage library (using e.g. m13, fd, or lambda phage),displaying inserts from 4 to about 80 amino acid residues usingconventional procedures. The inserts may represent, for example, acompletely degenerate or biased array. One then can select phage-bearinginserts which bind to the human N-type calcium channel hα_(1B+SFVG)subunit polypeptide. This process can be repeated through several cyclesof reselection of phage that bind to the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. Repeated rounds lead to enrichment ofphage bearing particular sequences. DNA sequence analysis can beconducted to identify the sequences of the expressed polypeptides. Theminimal linear portion of the sequence that binds to the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptides. Thus, the human N-type calcium channel hα_(1B+SFVG)subunit polypeptides of the invention, or a fragment thereof, can beused to screen peptide libraries, including phage display libraries, toidentify and select peptide binding partners of the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides of the invention. Suchmolecules can be used, as described, for screening assays, forpurification protocols, for interfering directly with the functioning ofhuman N-type calcium channel hα_(1B+SFVG) subunit and for other purposesthat will be apparent to those of ordinary skill in the art.

[0095] Peptides may easily be synthesized or produced by recombinantmeans by those of skill in the art. Using routine procedures known tothose of ordinary skill in the art, one can determine whether a peptidewhich binds to human N-type calcium channel hα_(1B+SFVG) subunit isuseful according to the invention by determining whether the peptide isone which inhibits the activity of human N-type calcium channelhα_(1B+SFVG) subunit in a voltage gated calcium influx assay, asdiscussed above.

[0096] The human N-type calcium channel hα_(1B+SFVG) subunit bindingpeptide agent may also be an antibody or a functionally active antibodyfragment. Antibodies are well known to those of ordinary skill in thescience of immunology. As used herein, the term “antibody” means notonly intact antibody molecules but also fragments of antibody moleculesretaining human N-type calcium channel hα_(1B+SFVG) subunit bindingability. Such fragments are also well known in the art and are regularlyemployed both in vitro and in vivo. In particular, as used herein, theterm “antibody” means not only intact immunoglobulin molecules but alsothe well-known active fragments F(ab′)₂, and Fab. F(ab′)₂, and Fabfragments which lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)).

[0097] In one set of embodiments, the antibody useful according to themethods of the present invention is an intact, fully human anti-humanN-type calcium channel hα_(1B+SFVG) subunit monoclonal antibody in anisolated form or in a pharmaceutical preparation. The following is adescription of a method for developing a monoclonal antibody thatinteracts with and inhibits the activity of human N-type calcium channelhα_(1B+SFVG) subunit. The description is exemplary and is provided forillustrative purposes only.

[0098] Murine monoclonal antibodies may be made by any of the methodsknown in the art utilizing human N-type calcium channel hα_(1B+SFVG)subunit, or a fragment thereof, as an immunogen provided that thechannel or fragment contains the amino acid sequence of SEQ ID NO: 2.

[0099] Human monoclonal antibodies may be made by any of the methodsknown in the art, such as those disclosed in U.S. Pat. No. 5,567,610,issued to Borrebaeck et al., U.S. Pat. No. 5,565,354, issued to Ostberg,U.S. Pat. No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133:3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniquesand Applications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), andBoerner et al., J. Immunol., 147: 86-95 (1991). In addition to theconventional methods for preparing human monoclonal antibodies, suchantibodies may also be prepared by immunizing transgenic animals thatare capable of producing human antibodies (e.g., Jakobovits et al.,Proc. Nat'l. Acad. Sci. USA, 90: 2551 (1993), Jakobovits et al., Nature,362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 (1993)and U.S. Pat. No. 5,569,825 issued to Lonberg).

[0100] Alternatively the antibody may be a polyclonal antibody specificfor human N-type calcium channel hα_(1B+SFVG) subunit which inhibitshuman N-type calcium channel hα_(1B+SFVG) subunit activity. Thepreparation and use of polyclonal antibodies is known to one of ordinaryskill in the art.

[0101] Significantly, as is well known in the art, only a small portionof an antibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

[0102] Within the antigen-binding portion of an antibody, as iswell-known in the art, there are complementarity determining regions(CDRs), which directly interact with the epitope of the antigen, andframework regions (FRs), which maintain the tertiary structure of theparatope (see, in general, Clark, 1986; Roitt, 1991). In both the heavychain Fd fragment and the light chain of IgG immunoglobulins, there arefour framework regions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

[0103] In general, intact antibodies are said to contain “Fe” and “Fab”regions. The Fc regions are involved in complement activation and arenot involved in antigen binding. An antibody from which the Fc′ regionhas been enzymatically cleaved, or which has been produced without theFc′ region, designated an “F(ab′)₂” fragment, retains both of theantigen binding sites of the intact antibody. Similarly, an antibodyfrom which the Fc region has been enzymatically cleaved, or which hasbeen produced without the Fc region, designated an “Fab′” fragment,retains one of the antigen binding sites of the intact antibody. Fab′fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain, denoted “Fd.” The Fd fragments arethe major determinants of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity). Isolated Fd fragments retain the ability tospecifically bind to antigen epitopes.

[0104] The sequences of the antigen-binding Fab′ portion of theanti-human N-type calcium channel hα_(1B+SFVG) subunit monoclonalantibodies identified as being useful according to the invention in theassays provided above, as well as the relevant FR and CDR regions, canbe determined using amino acid sequencing methods that are routine inthe art. It is well established that non-CDR regions of a mammalianantibody may be replaced with corresponding regions of non-specific orhetero-specific antibodies while retaining the epitope specificity ofthe original antibody. This technique is useful for the development anduse of “humanized” antibodies in which non-human CDRs are covalentlyjoined to human FR and/or Fc/pFc′ regions to produce a functionalantibody. Techniques to humanize antibodies are particularly useful whennon-human animal (e.g., murine) antibodies which inhibit human N-typecalcium channel hα_(1B+SFVG) subunit activity are identified. Thesenon-human animal antibodies can be humanized for use in the treatment ofa human subject in the methods according to the invention. An example ofa method for humanizing a murine antibody is provided in PCTInternational Publication No. WO 92/04381 which teaches the productionand use of humanized murine RSV antibodies in which at least a portionof the murine FR regions have been replaced by FR regions of humanorigin. Such antibodies, including fragments of intact antibodies withantigen-binding ability, are often referred to as “chimeric” antibodies.

[0105] Thus, as will be apparent to one of ordinary skill in the art,the present invention also provides for F(ab′)₂, and Fab fragments of ananti-human N-type calcium channel hα_(1B+SFVG) subunit monoclonalantibody; chimeric antibodies in which the Fc and/or FR and/or CDR1and/or CDR2 and/or light chain CDR3 regions of an anti-human N-typecalcium channel hα_(1B+SFVG) subunit antibody have been replaced byhomologous human or non-human sequences; chimeric F(ab′)₂ fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or light chainCDR3 regions of an anti-human N-type calcium channel hα_(1B+SFVG)subunit antibody have been replaced by homologous human or non-humansequences; and chimeric Fab fragment antibodies in which the FR and/orCDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced byhomologous human or non-human sequences. Thus, those skilled in the artmay alter an anti-human N-type calcium channel hα_(1B+SFVG) subunitantibody by the construction of CDR grafted or chimeric antibodies orantibody fragments containing, all or part thereof, of the disclosedheavy and light chain V-region CDR amino acid sequences (Jones et al.,Nature 321:522, 1986; Verhoeyen et al., Science 39:1534, 1988 andTempest et al., Bio/Technology 9:266, 1991), without destroying thespecificity of the antibodies for human N-type calcium channelhα_(1B+SFVG) subunit. Such CDR grafted or chimeric antibodies orantibody fragments can be effective in inhibiting human N-type calciumchannel hα_(1B+SFVG) subunit activity in animals (e.g. primates) andhumans.

[0106] In preferred embodiments, the chimeric antibodies of theinvention are fully human monoclonal antibodies. As noted above, suchchimeric antibodies may be produced in which some or all of the FRregions of human N-type calcium channel hα_(1B+SFVG) subunit have beenreplaced by other homologous human FR regions. In addition, the Feportions may be replaced so as to produce IgA or IgM as well as IgGantibodies bearing some or all of the CDRs of the anti-human N-typecalcium channel hα_(1B+SFVG) subunit antibody. Of particular importanceis the inclusion of the anti-human N-type calcium channel hα_(1B+SFVG)subunit heavy chain CDR3 region and, to a lesser extent, the other CDRsof anti-human N-type calcium channel hα_(1B+SFVG) subunit antibodies.Such fully human chimeric antibodies will have particular utility inthat they will not evoke an immune response against the antibody itself.

[0107] It is also possible, in accordance with the present invention, toproduce chimeric antibodies including non-human sequences. Thus, one mayuse, for example, murine, ovine, equine, bovine or other mammalian Fc orFR sequences to replace some or all of the Fc or FR regions of theanti-human N-type calcium channel hα_(1B+SFVG) subunit antibody. Some ofthe CDRs may be replaced as well. Again, however, it is preferred thatat least the heavy chain CDR3 region of the anti-human N-type calciumchannel hα_(1B+SFVG) subunit antibody be included in such chimericantibodies and, to a lesser extent, it is also preferred that some orall of the other CDRs of anti-human N-type calcium channel hα_(1B+SFVG)subunit be included. Such chimeric antibodies bearing non-humanimmunoglobulin sequences admixed with the CDRs of the human anti-humanN-type calcium channel hα_(1B+SFVG) subunit monoclonal antibody are notpreferred for use in humans and are particularly not preferred forextended use because they may evoke an immune response against thenon-human sequences. They may, of course, be used for brief periods orin immunosuppressed individuals but, again, fully human antibodies arepreferred. Because, however, such antibodies may be used for briefperiods or in immunosuppressed subjects, chimeric antibodies bearingnon-human mammalian Fc and FR sequences but including at least theanti-human N-type calcium channel hα_(1B+SFVG) subunit heavy chain CDR3are contemplated as alternative embodiments of the present invention.

[0108] For inoculation or prophylactic uses, the antibodies of thepresent invention are preferably intact antibody molecules including theFc region. Such intact antibodies will have longer half-lives thansmaller fragment antibodies (e.g. Fab) and are more suitable forintravenous, intraperitoneal, intramuscular, intracavity, subcutaneous,or transdermal administration.

[0109] Fab fragments, including chimeric Fab fragments, are preferred inmethods in which the antibodies of the invention are administereddirectly to a local tissue environment. For example, the Fab fragmentsare preferred when the antibody of the invention is administereddirectly to the brain. Fabs offer several advantages over F(ab′)₂ andwhole immunoglobulin molecules for this therapeutic modality. First,because Fabs have only one binding site for their cognate antigen, theformation of immune complexes is precluded whereas such complexes can begenerated when bivalent F(ab′)₂ s and whole immunoglobulin moleculesencounter their target antigen. This is of some importance becauseimmune complex deposition in tissues can produce adverse inflammatoryreactions. Second, because Fabs lack an Fc region they cannot triggeradverse inflammatory reactions that are activated by Fc, such asactivation of the complement cascade. Third, the tissue penetration ofthe small Fab molecule is likely to be much better than that of thelarger whole antibody. Fourth, Fabs can be produced easily andinexpensively in bacteria, such as E. coli, whereas whole immunoglobulinantibody molecules require mammalian cells for their production inuseful amounts. Production of Fabs in E. coli makes it possible toproduce these antibody fragments in large fermenters which are lessexpensive than cell culture-derived products.

[0110] Smaller antibody fragments and small binding peptides havingbinding specificity for the human N-type calcium channel hα_(1B+SFVG)subunit which can be used to inhibit human N-type calcium channelhα_(1B+SFVG) subunit activity also are embraced within the presentinvention. For example, single-chain antibodies can be constructed inaccordance with the methods described in U.S. Pat. No. 4,946,778 toLadner et al. Such single-chain antibodies include the variable regionsof the light and heavy chains joined by a flexible linker moiety.Methods for obtaining a single domain antibody (“Fd”) which comprises anisolated VH single domain, also have been reported (see, for example,Ward et al., Nature 341:644-646 (1989)).

[0111] According to the invention human N-type calcium channelhα_(1B+SFVG) subunit inhibitors also include “dominant negative”polypeptides derived from SEQ ID NO: 4. A dominant negative polypeptideis an inactive variant of a polypeptide, which, by interacting with thecellular machinery, displaces an active polypeptide from its interactionwith the cellular machinery or competes with the active polypeptide,thereby reducing the effect of the active polypeptide. For example, adominant negative receptor which binds a ligand but does not transmit asignal in response to binding of the ligand can reduce the biologicaleffect of expression of the ligand. Likewise, a dominant negative humanN-type calcium channel hα_(1B+SFVG) subunit of an active complex (e.g.N-type calcium channel) can interact with the complex but prevent theactivity of the complex (e.g. voltage gated calcium influx).

[0112] The end result of the expression of a dominant negative humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide of the inventionin a cell is a reduction in voltage gated calcium influx. One ofordinary skill in the art can assess the potential for a dominantnegative variant of a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide, and using standard mutagenesis techniques to create one ormore dominant negative variant polypeptides. For example, given theteachings contained herein of a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide, one of ordinary skill in the art canmodify the sequence of the human N-type calcium channel hα_(1+SFVG)subunit polypeptide by site-specific mutagenesis, scanning mutagenesis,partial gene deletion or truncation, and the like. See, e.g., U.S. Pat.No. 5,580,723 and Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Theskilled artisan then can test the population of mutagenized polypeptidesfor diminution in human N-type calcium channel hα_(1B+SFVG) subunitactivity (e.g., voltage gated calcium influx) and/or for retention ofsuch an activity. Other similar methods for creating and testingdominant negative variants of a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide will be apparent to one of ordinaryskill in the art.

[0113] Each of the compositions of the invention is useful for a varietyof therapeutic and non-therapeutic purposes. For example, the humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acids of theinvention are useful as oligonucleotide probes. Such oligonucleotideprobes can be used herein to identify genomic or cDNA library clonespossessing an identical or substantially similar nucleic acid sequence.A suitable oligonucleotide or set of oligonucleotides, which is capableof hybridizing under stringent hybridization conditions to the desiredsequence, a variant or fragment thereof, or an anti-sense complement ofsuch an oligonucleotide or set of oligonucleotides, can be synthesizedby means well known in the art (see, for example, Synthesis andApplication of DNA and RNA, S. A. Narang, ed., 1987, Academic Press, SanDiego, Calif.) and employed as a probe to identify and isolate thedesired sequence, variant or fragment thereof by techniques known in theart. Techniques of nucleic acid hybridization and clone identificationare disclosed by Sambrook, et al., Molecular Cloning, A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989), and by Hames, B. D., et al., in Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, D.C. (1985). To facilitatethe detection of a desired nucleic acid sequence, or variant or fragmentthereof, whether for cloning purposes or for the mere detection of thepresence of the sequence, the above-described probes may be labeled witha detectable group. Such a detectable group may be any material having adetectable physical or chemical property. Such materials have beenwell-developed in the field of nucleic acid hybridization and, ingeneral, most any label useful in such methods can be applied to thepresent invention.

[0114] Particularly useful are radioactive labels. Any radioactive labelmay be employed which provides for an adequate signal and has asufficient half-life. If single stranded, the oligonucleotide may beradioactively labeled using kinase reactions. Alternatively,oligonucleotides are also useful as nucleic acid hybridization probeswhen labeled with a non-radioactive marker such as biotin, an enzyme ora fluorescent group. See, for example, Leary, J. J., et al., Proc. Natl.Acad. Sci. (USA) 80:4045 (1983); Renz, M. et al., Nucl. Acids Res.12:3435 (1984); and Renz, M., EMBO J. 6:817 (1983).

[0115] Additionally, complements of the human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids can be useful as antisenseoligonucleotides, e.g., by delivering the antisense oligonucleotide toan animal to induce a human N-type calcium channel hα_(1B+SFVG) subunit“knockout” phenotype. The administration of antisense RNA probes toblock gene expression is discussed in Lichtenstein, C., Nature333:801-802 (1988).

[0116] Alternatively, the human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid of the invention can be used to prepare a non-humantransgenic animal. A “transgenic animal” is an animal having cells thatcontain DNA which has been artificially inserted into a cell, which DNAbecomes part of the genome of the animal which develops from that cell.Preferred transgenic animals are primates, mice, rats, cows, pigs,horses, goats, sheep, dogs and cats. Animals suitable for transgenicexperiments can be obtained from standard commercial sources such asCharles River (Wilmington, Mass.), Taconic (Germantown, N.Y.), HarlanSprague Dawley (Indianapolis, Ind.), etc. Transgenic animals having aparticular property associated with a particular disease can be used tostudy the affects of a variety of drugs and treatment methods on thedisease, and thus serve as genetic models for the study of a number ofhuman diseases. The invention, therefore, contemplates the use of humanN-type calcium channel hα_(1B+SFVG) subunit knockout and transgenicanimals as models for the study of disorders involving voltage gatedcalcium influx.

[0117] A variety of methods are available for the production oftransgenic animals associated with this invention. DNA can be injectedinto the pronucleus of a fertilized egg before fusion of the male andfemale pronuclei, or injected into the nucleus of an embryonic cell(e.g., the nucleus of a two-cell embryo) following the initiation ofcell division. See e.g., Brinster et al., Proc. Nat. Acad. Sci. USA, 82:4438 (1985); Brinster et al., Cell 27: 223 (1981); Costantini et al.,Nature 294: 982 (1981); Harpers et al., Nature 293: 540 (1981); Wagneret al., Proc. Nat. Acad. Sci. USA 78:5016 (1981); Gordon et al., Proc.Nat. Acad. Sci. USA 73: 1260 (1976). The fertilized egg is thenimplanted into the uterus of the recipient female and allowed to developinto an animal.

[0118] An alternative method for producing transgenic animals involvesthe incorporation of the desired gene sequence into a virus which iscapable of affecting the cells of a host animal. See e.g., Elbrecht etal., Molec. Cell. Biol. 7: 1276 (1987); Lacey et al., Nature 322: 609(1986); Leopol et al., Cell 51: 885 (1987). Embryos can be infected withviruses, especially retroviruses, modified to carry the nucleotidesequences of the invention which encode human N-type calcium channelhα_(1B+SFVG) subunit proteins or sequences which disrupt the nativehuman N-type calcium channel hα_(1B+SFVG) subunit gene to produce aknockout animal.

[0119] Another method for producing transgenic animals involves theinjection of pluripotent embryonic stem cells into a blastocyst of adeveloping embryo. Pluripotent stem cells derived from the inner cellmass of the embryo and stabilized in culture can be manipulated inculture to incorporate nucleotide sequences of the invention. Atransgenic animal can be produced from such cells through implantationinto a blastocyst that is implanted into a foster mother and allowed tocome to term. See e.g., Robertson et al., Cold Spring Harbor ConferenceCell Proliferation 10: 647 (1983); Bradley et al., Nature 309: 255(1984); Wagner et al., Cold Spring Harbor Symposium Quantitative Biology50: 691 (1985).

[0120] The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia, 47: 897-905 (1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

[0121] By way of example only, to prepare a transgenic mouse, femalemice are induced to superovulate. Females are placed with males, and themated females are sacrificed by CO₂ asphyxiation or cervical dislocationand embryos are recovered from excised oviducts. Surrounding cumuluscells are removed. Pronuclear embryos are then washed and stored untilthe time of injection. Randomly cycling adult female mice are pairedwith vasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer etal., Cell, 63:1099-1112 (1990).

[0122] Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as etectroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press (1987).

[0123] In cases involving random gene integration, a clone containingthe sequence(s) of the invention is co-transfected with a gene encodingresistance. Alternatively, the gene encoding neomycin resistance isphysically linked to the sequence(s) of the invention. Transfection andisolation of desired clones are carried out by any one of severalmethods well known to those of ordinary skill in the art (E. J.Robertson, supra).

[0124] DNA molecules introduced into ES cells can also be integratedinto the chromosome through the process of homologous recombination.Capecchi, Science, 244: 1288-1292 (1989). Methods for positive selectionof the recombination event (e.g., neo resistance) and dualpositive-negative selection (e.g., neo resistance and gancyclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Capecchi, supra and Joyner et al., Nature,338: 153-156 (1989). The final phase of the procedure is to injecttargeted ES cells into blastocysts and to transfer the blastocysts intopseudopregnant females. The resulting chimeric animals are bred and theoffspring are analyzed by Southern blotting to identify individuals thatcarry the transgene.

[0125] Procedures for the production of non-rodent mammals and otheranimals have been discussed by others. See Houdebine and Chourrout,supra; Pursel et al., Science 244: 1281-1288 (1989); and Simms et al.,Bio/Technology, 6: 179-183 (1988).

[0126] Inactivation or replacement of the endogenous N-type calciumchannel hα_(1B+SFVG) subunit gene can be achieved by a homologousrecombination system using embryonic stem cells. The resultanttransgenic non-human mammals (preferably primates) having a knockoutN-type calcium channel hα_(1B+SFVG) subunit characteristic may be madetransgenic for the human N-type calcium channel hα_(1B+SFVG) subunit andused as a model for screening compounds as modulators (agonists orantagonists/inhibitors) of the human N-type calcium channel hα_(1B+SFVG)subunit. In this manner, such therapeutic drugs can be identified.

[0127] Additionally, a normal or mutant version of human N-type calciumchannel hα_(1B+SFVG) subunit can be inserted into the mouse (or theanimal) germ line to produce transgenic animals which constitutively orinducibly express the normal or mutant form of human N-type calciumchannel hα_(1B+SFVG) subunit. These animals are useful in studies todefine the role and function of human N-type calcium channelhα_(1B+SFVG) subunit in cells. These studies are particularly useful inanimals, which do not normally express human N-type calcium channelhα_(1B+SFVG) subunit, such as non-primates.

[0128] A human N-type calcium channel hα_(1B+SFVG) subunit polypeptide,or a fragment thereof, also can be used to isolate human N-type calciumchannel hα_(1B+SFVG) subunit native binding partners, including, e.g.,the N-type calcium channel. Isolation of such binding partners may beperformed according to well-known methods. For example, isolated humanN-type calcium channel hα_(1B+SFVG) subunit polypeptides can be attachedto a substrate (e.g., chromatographic media, such as polystyrene beads,or a filter), and then a solution suspected of containing the N-typecalcium channel may be applied to the substrate. If a N-type calciumchannel which can interact with human N-type calcium channelhα_(1B+SFVG) subunit polypeptides is present in the solution, then itwill bind to the substrate-bound human N-type calcium channelhα_(1B+SFVG) subunit polypeptide. The N-type calcium channel then may beisolated. Other polypeptides which are binding partners for human N-typecalcium channel hα_(1B+SFVG) subunit may be isolated by similar methodswithout undue experimentation.

[0129] The compositions of the invention are also useful for therapeuticpurposes. Accordingly the invention encompasses a method for inhibitinghuman N-type calcium channel hα_(1B+SFVG) subunit activity in amammalian cell. The invention further provides methods for reducing orincreasing human N-type calcium channel hα_(1B+SFVG) subunit activity ina cell. In one embodiment, the method involves contacting the mammaliancell with an amount of a human N-type calcium channel hα_(1B+SFVG)subunit inhibitor effective to inhibit voltage gated calcium influx inthe mammalian cell. Such methods are useful in vitro for alteringvoltage gated calcium influx for the purpose of, for example,elucidating the mechanisms involved in stroke, pain, e.g., neuropathicpain, and traumatic brain injury and for restoring the voltage gatedcalcium influx in a cell having a defective human N-type calcium channelhα_(1B+SFVG) subunit. In vivo, such methods are useful, for example, forreducing N-type voltage gated calcium influx, e.g., to treat stroke,pain, e.g., neuropathic pain, traumatic brain injury, or any conditionin which human N-type calcium channel hα_(1B+SFVG) subunit activity iselevated.

[0130] An amount of a human N-type calcium channel hα_(1B+SFVG) subunitinhibitor which is effective to inhibit voltage gated calcium influx inthe mammalian cell is an amount which is sufficient to reduce voltagegated calcium influx by at least 10%, preferably at least 20%, morepreferably 30% and still more preferably 40%. An amount of a humanN-type calcium channel hα_(1B+SFVG) subunit which is effective toincrease voltage gated calcium influx in the mammalian cell is an amountwhich is sufficient to increase voltage gated calcium influx by at least10%, preferably at least 20%, more preferably 30% and still morepreferably 40%. Such alterations in voltage gated calcium influx can bemeasured by the assays described herein.

[0131] As described above with respect to inhibitors, modulators ofhα_(1B+SFVG) may selectively inhibit or increase hα_(1B+SFVG) functionbased on the state of depolarization of the membrane with which thehα_(1B+SFVG) is associated. Therefore, in screening for modulators ofhα_(1B+SFVG) it is preferred that compounds (e.g. syntheticcombinatorial libraries, natural products, peptide libraries, etc.) aretested for modulation of hα_(1B+SFVG) activity at a variety of voltageswhich cause partial or complete membrane depolarization, orhyperpolarization. These assays are conducted according to standardprocedures of testing calcium channel function (e.g. patch clamping,fluorescent Ca²⁺ influx assays) which require no more than routineexperimentation. Using such methods, modulators of hα_(1B+SFVG) activitywhich are active at particular voltages (e.g. complete membranedepolarization) can be identified. Such compounds are useful forselectively modulating calcium channel activity in conditions which maydisplay voltage dependence.

[0132] The invention also encompasses a method for increasing humanN-type calcium channel hα_(1B+SFVG) subunit expression in a cell orsubject. It is desirable to increase human N-type calcium channelhα_(1B+SFVG) subunit in a subject that has a disorder characterized by adeficiency in voltage gated calcium influx. The amount of human N-typecalcium channel hα_(1B+SFVG) subunit can be increased in such cell orsubject by contacting the cell with, or administering to the subject, ahuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid or ahuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide of theinvention to the subject in an amount effective to increase voltagegated calcium influx in the cell or the subject. An increase in humanN-type calcium channel hα_(1B+SFVG) subunit activity can be measured bythe assays described herein, e.g., assays of calcium influx.

[0133] The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.

[0134] The preparations of the invention are administered in effectiveamounts. An effective amount is that amount of a pharmaceuticalpreparation that alone, or together with further doses, produces thedesired response. In the case of treating a condition characterized byaberrant voltage gated calcium influx, the desired response is reducingor increasing calcium influx to a level which is within a normal range.Preferably, the change in calcium influx produces a detectable reductionin a physiological function related to the condition, e.g., a reductionin neurotoxicity following stroke. The responses can be monitored byroutine methods. In the case of a condition where an increase in voltagegated calcium influx is desired, an effective amount is that amountnecessary to increase said influx in the target tissue. The converse isthe case when a reduction in influx is desired. An increase or decreasein neurotransmitter release also could be measured to monitor theresponse.

[0135] Such amounts will depend, of course, on the particular conditionbeing treated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. It is preferredgenerally that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons.

[0136] Generally, doses of active compounds would be from about 0.01mg/kg per day to 1000 mg/kg per day. It is expected that doses rangingfrom 50-500 mg/kg will be suitable and in one or several administrationsper day. Lower doses will result from other forms of administration,such as intravenous administration. In the event that a response in asubject is insufficient at the initial doses applied, higher doses (oreffectively higher doses by a different, more localized delivery route)may be employed to the extent that patient tolerance permits. Multipledoses per day are contemplated to achieve appropriate systemic levels ofcompound, although fewer doses typically will be given when compoundsare prepared as slow release or sustained release medications.

[0137] When administered, the pharmaceutical preparations of theinvention are applied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salts, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

[0138] The human N-type calcium channel hα_(1B+SFVG) subunit inhibitorsor human N-type calcium channel hα_(1B+SFVG) subunit nucleic acids andpolypeptides useful according to the invention may be combined,optionally, with a pharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

[0139] The pharmaceutical compositions may contain suitable bufferingagents, including: acetic acid in a salt; citric acid in a salt; andphosphoric acid in a salt.

[0140] The pharmaceutical compositions also may contain, optionally,suitable preservatives, such as: benzalkonium chloride; chlorobutanol;parabens and thimerosal.

[0141] A variety of administration routes are available. The particularmode selected will depend, of course, upon the particular compoundselected, the severity of the condition being treated and the dosagerequired for therapeutic efficacy. The methods of the invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, nasal, interdermal, or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, intrathecal, intramuscular, orinfusion. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis.

[0142] The pharmaceutical compositions may conveniently be presented inunit dosage form and may be prepared by any of the methods well-known inthe art of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

[0143] Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

[0144] Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the human N-type calciumchannel hα_(1B+SFVG) subunit inhibitor or human N-type calcium channelhα_(1B+SFVG) subunit nucleic acids and polypeptides, which is preferablyisotonic with the blood of the recipient. This aqueous preparation maybe formulated according to known methods using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationalso may be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,3-butane diol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intrathecal, intramuscular, etc. administrations can befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

[0145] Other delivery systems can include time-release, delayed releaseor sustained release delivery systems such as the biological/chemicalvectors is discussed above. Such systems can avoid repeatedadministrations of the active compound, increasing convenience to thesubject and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. Use of along-term sustained release implant may be desirable. Long-term release,are used herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

[0146] The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents useful in thetreatment of conditions associated with aberrant voltage gated cellcalcium influx mediated by human N-type calcium channel hα_(1B+SFVG)subunit and the compounds and agents so identified. Generally, thescreening methods involve assaying for compounds which inhibit orenhance voltage gated calcium influx through human N-type calciumchannels. Such methods are adaptable to automated, high throughputscreening of compounds. Examples of such methods are described in U.S.Pat. No. 5,429,921.

[0147] A variety of assays for pharmacological agents are provided,including, labeled in vitro protein binding assays, Ca²⁺influx assays,etc. For example, protein binding screens are used to rapidly examinethe binding of candidate pharmacological agents to a human N-typecalcium channel hα_(1B+SFVG) subunit. The candidate pharmacologicalagents can be derived from, for example, combinatorial peptidelibraries. Convenient reagents for such assays are known in the art. Anexemplary cell-based assay of calcium influx involves contacting aneuronal cell having a human N-type calcium channel hα_(1B+SFVG) subunitwith a candidate pharmacological agent under conditions whereby theinflux of calcium can be stimulated by application of a voltage to thetest system, i.e., by membrane depolarization. Specific conditions arewell known in the art and are described in Lin et al., Neuron18:153-166, 1997, and in U.S. Pat. No. 5,429,921. A reduction in thevoltage gated calcium influx in the presence of the candidatepharmacological agent indicates that the candidate pharmacological agentreduces the induction of calcium influx of human N-type calcium channelhα_(1B+SFVG) subunit in response to the voltage stimulus. An increase inthe voltage gated calcium influx in the presence of the candidatepharmacological agent indicates that the candidate pharmacological agentincreases the induction of calcium influx of human N-type calciumchannel hα_(1B+SFVG) subunit in response to the voltage stimulus.Methods for determining changes in the intracellular calciumconcentration are known in the art and are addressed elsewhere herein.

[0148] Human N-type calcium channel hα_(1B+SFVG) subunit used in themethods of the invention can be added to an assay mixture as an isolatedpolypeptide (where binding of a candidate pharmaceutical agent is to bemeasured) or as a cell or other membrane-encapsulated space whichincludes a human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide. In the latter assay configuration, the cell or othermembrane-encapsulated space can contain the human N-type calcium channelhα_(1B+SFVG) subunit as a preloaded polypeptide or as a nucleic acid(e.g. a cell transfected with an expression vector containing a humanN-type calcium channel hα_(1B+SFVG) subunit). In the assays describedherein, the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide can be produced recombinantly, or isolated from biologicalextracts, but preferably is synthesized in vitro. Human N-type calciumchannel hα_(1B+SFVG) subunit polypeptides encompass chimeric proteinscomprising a fusion of a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide with another polypeptide, e.g., a polypeptidecapable of providing or enhancing protein-protein binding, or enhancingstability of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide under assay conditions. A polypeptide fused to a humanN-type calcium channel hα_(1B+SFVG) subunit polypeptide or fragmentthereof may also provide means of readily detecting the fusion protein,e.g., by immunological recognition or by fluorescent labeling.

[0149] The assay mixture also comprises a candidate pharmacologicalagent. Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate pharmacological agents aresmall organic compounds, i.e., those having a molecular weight of morethan 50 yet less than about 2500. Candidate agents comprise functionalchemical groups necessary for structural interactions with polypeptides,and typically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as peptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is anucleic acid, the agent typically is a DNA or RNA molecule, althoughmodified nucleic acids having non-natural bonds or subunits are alsocontemplated.

[0150] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

[0151] Candidate agents can be selected randomly or can be based onexisting compounds which bind to and/or modulate the function of N-typecalcium channels. For example, compounds which are known to inhibitN-type calcium channels include fluspirilene, ziconotide (SNX-111), theω-conotoxin peptides GVIA (SEQ ID NO: 11) and MVIIA (SEQ ID NO: 12), aswell as small organic molecule calcium channel inhibitors, such asfluspirilene, NNC09-0026(−)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-phenoxy)methyl] piperidinedihydrochloride); SB 201823-A(4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidinehydrochloride); NS649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole);CNS 1237 (N-acenaphthyl-N′-4-methoxynaphth-1-yl guanidine) and riluzole.Therefore, a source of candidate agents are libraries of molecules basedon the foregoing N-type calcium channel inhibitors, in which thestructure of the inhibitor is changed at one or more positions of themolecule to contain more or fewer chemical moieties or differentchemical moieties. The structural changes made to the molecules increating the libraries of analog inhibitors can be directed, random, ora combination of both directed and random substitutions and/oradditions. One of ordinary skill in the art in the preparation ofcombinatorial libraries can readily prepare such libraries based on theexisting N-type calcium channel inhibitors.

[0152] A variety of other reagents also can be included in the mixture.These include reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

[0153] The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, the human N-type calcium channel hα_(1B+SFVG)subunit transduces a control amount of voltage gated calcium influx. Fordetermining the binding of a candidate pharmaceutical agent to a humanN-type calcium channel hα_(1B+SFVG) subunit, the mixture is incubatedunder conditions which permit binding. The order of addition ofcomponents, incubation temperature, time of incubation, and otherparameters of the assay may be readily determined. Such experimentationmerely involves optimization of the assay parameters, not thefundamental composition of the assay. Incubation temperatures typicallyare between 4° C. and 40° C. Incubation times preferably are minimizedto facilitate rapid, high throughput screening, and typically arebetween 1 minute and 10 hours.

[0154] After incubation, the level of voltage gated calcium influx orthe level of specific binding between the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide and the candidate pharmaceutical agentis detected by any convenient method available to the user. For cellfree binding type assays, a separation step is often used to separatebound from unbound components. The separation step may be accomplishedin a variety of ways. Conveniently, at least one of the components isimmobilized on a solid substrate, from which the unbound components maybe easily separated. The solid substrate can be made of a wide varietyof materials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximize signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

[0155] Separation may be effected for example, by removing a bead ordipstick from a reservoir, emptying or diluting a reservoir such as amicrotiter plate well, rinsing a bead, particle, chromatographic columnor filter with a wash solution or solvent. The separation steppreferably includes multiple rinses or washes. For example, when thesolid substrate is a microtiter plate, the wells may be washed severaltimes with a washing solution, which typically includes those componentsof the incubation mixture that do not participate in specific bindingssuch as salts, buffer, detergent, non-specific protein, etc. Where thesolid substrate is a magnetic bead, the beads may be washed one or moretimes with a washing solution and isolated using a magnet.

[0156] Detection may be effected in any convenient way for cell-basedassays such as a calcium influx assay. The calcium influx resulting fromvoltage stimulus of the human N-type calcium channel hα_(1B+SFVG)subunit polypeptide typically alters a directly or indirectly detectableproduct, e.g., a calcium sensitive molecule such as fura-2-AM. For cellfree binding assays, one of the components usually comprises, or iscoupled to, a detectable label. A wide variety of labels can be used,such as those that provide direct detection (e.g., radioactivity,luminescence, optical or electron density, etc). or indirect detection(e.g., epitope tag such as the FLAG epitope, enzyme tag such ashorseradish peroxidase, etc.). The label may be bound to a human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide or the candidatepharmacological agent.

[0157] A variety of methods may be used to detect the label, dependingon the nature of the label and other assay components. For example, thelabel may be detected while bound to the solid substrate or subsequentto separation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

[0158] The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1

[0159] Analysis of human brain N-type calcium channel splice variants

[0160] The abundance of splice variants of N-type calcium channels inhuman brain was determined using polymerase chain reaction analysis andRNase protection assays as described in Lin et al. (Neuron 18:153-166,1997). Human N-type calcium channel α_(1B) subunit clones were sequencedby standard methods of nucleotide sequencing and it was determined thatone type of clone had a 12 nucleotide insert (SEQ ID NO: 1) as comparedto previously published human N-type calcium channel α_(1B) subunitsequences. The present human N-type calcium channel α_(1B) subunitnucleic acid molecule (designated hα_(1B+SFVG)) corresponds to thepublished nucleotide sequence for human N-type calcium channel α_(1B)subunits with the 12 nucleotide insert located after nucleotide 3855 (asnumbered in Williams et al., Science 257:389-395, 1992). The nucleotidesequence of SEQ ID NO: 1 supplies the third base of the codon encodingSer1237, three new codons (Ser1238, Phe1239 and Val1240), and the firsttwo bases of codon Gly1241, as shown in SEQ ID NO: 3: tcG AGC TTC GTGGGa (insert in caps). This insert thus encodes a four amino acid insertin the protein which is similar to, but surprisingly is not identicalto, amino acids 1236-1239 of a rat N-type calcium channel α_(1B) subunit(SEQ ID NO: 10, GenBank accession number M92905). The human N-typecalcium channel hα_(1B+SFVG) subunit was found to make up a significantportion of the N-type calcium channel α_(1B) subunits mRNA in humanbrain. It was also determined that the hα_(1B+SFVG) subunit wasdifferentially distributed in different parts of the brain, e.g. incertain portions of the brain hα_(1B+SFVG) was more highly expressedthan in other portions of the brain.

Example 2

[0161] Construction of Human N-type Calcium Channel hα_(1B+SFVG) SubunitNucleic Acids

[0162] The human N-type calcium channel hα_(1B+SFVG) subunit containingthe SFVG insert is constructed according to standard proceduresdescribed in, e.g., Current Protocols in Molecular Biology (F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York), using PCRprimers which contain the nucleotides encoding SFVG (e.g., SEQ ID NO: 1)to amplify the published human N-type calcium channel c,B subunitnucleic acid. Fragments generated by PCR are then assembled by ligationto prepare a complete cDNA encoding the human hα_(1B+SFVG) subunit.

Example 3

[0163] Function of the Human N-type Calcium Channel hα_(1B+SFVG) Subunit

[0164] The voltage gated calcium channel activity of the human N-typecalcium channel hα_(1B+SFVG) subunit is tested using to the methodsdescribed in Lin et al. (1997) for a rat N-type calcium channel subunit,and as described in Example 4 below.

Example 4

[0165] Activation Differences in Rat N-type Calcium Channels ±the ETExon

[0166] Functional Assessment of the Calcium Channel α_(1B) cDNAConstructs

[0167] The functional properties of all calcium (Ca) channel α_(1B) cDNAconstructs described in this paper were assessed in the Xenopus oocyteexpression system. All methods and procedures were essentially the sameas described in Lin et al. (1997). cRNAs were in vitro transcribed usingthe mMESSAGE mMACHINE kit (Ambion) from the various α_(1B) cDNAconstructs subcloned into the Xenopus β-globin expression vector (pBSTA;Goldin & Sumikawa et al., Methods EnzymoL 207:279-297, 1992). 46 nl of a750ng/μl cRNA solution was injected into defolliculated oocytes using aprecision nanoinjector (Drummond). N-type Ca channel currents wererecorded 6-7 days after injection. At least 15 minutes prior torecording, oocytes were injected with 46 nl of a 50 mM solution of BAPTA(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetate). This we havefound critical to minimize activation of an endogenous Ca²⁺-activatedCl⁻current, even when Ba²⁺is the charge carrier (Lin et al., 1997).Cells exhibiting slowly deactivating tail currents, indicative of thepresence of Ba²⁺-dependent activation of the Ca-activated Cl⁻ current,were excluded from the analysis.

[0168] N-type Ca²⁺channel currents were recorded from oocytes using thetwo microelectrode voltage-clamp recording technique (Warner amplifier;OC-725b). A virtual ground circuit eliminated the need for seriesresistance compensation when recording large currents. Micropipettes of0.8-1.5 MΩ and 0.3-0.5 MΩ resistance when filled with 3 M KCl were usedfor the voltage and current recording electrodes, respectively. Oocytesexpressing Ca²⁺channel currents usually had resting membrane potentialsbetween −40 and −50 mV when impaled with two electrodes. A groundedmetal shield was placed between the two electrodes to increase thesettling time of the clamp. Recording solutions contained 5 mM BaCl₂, 85mM tetraethylammonium, 5 mM KCl, and 5 mM HEPES (pH adjusted to 7.4 withmethanesulfonic acid). The recording temperature was between 19° C. and22° C.

[0169] The properties of each mutant construct were assessed byexpressing it together with appropriate controls (ΔET α_(1B) and +ETα_(1B)). Each mutant was tested in three separate batches of oocytes andwithin each batch, recordings were made from at least six oocytes foreach mutant construct and control. Recordings from the oocytesexpressing the various Ca channel (x,B constructs were randomizedthroughout the data collection period.

[0170] Data analysis

[0171] Data were acquired on-line and leak subtracted using a P/4protocol (PClamp V6.0; Axon Inst.). Voltage-steps were applied every10-30 seconds depending on the duration of the step, from a holdingpotential of −80 mV. Ca channel currents recorded under these conditionsshowed little run-down over the duration of the recordings. Three setsof current voltage-relationships were obtained from each cell using stepdepolarizations of 26.3 ms, 650 ms and 2.6 s in duration and digitizedat 25 kHz, 10 kHz and 250 Hz, respectively. Exponential curves(activation and inactivation) were fit to the data using curve fittingroutines in PClamp (Axon Instr.) and Origin (Microcal). Inactivationtime constants in the range of 70-800 msec were estimated from currentsevoked by the longest depolarization (2.6 s). Activation time constantswere best resolved from currents evoked by the shortest depolarizations(26.3 ms; sampled at 25 kHz).

[0172] Modeling Ca entry

[0173] A one-compartment cell model employing standard compartmentalmodeling techniques in NEURON (Hines & Carnevale, Neural Comput.9:1179-1209, 1997) was used to predict the amount of Ca entering aneuron expressing either rnα_(1B-b) or rnα_(1B-b) N-type Ca channelcurrents. The cell had a total membrane area of 1250 μm², 0.75 μF/cm²specific membrane capacitance and 30 kΩcm² specific membrane resistance.For action potential simulation a fast sodium conductance (g_(Na)) and adelayed rectifying potassium conductance (g_(K,Dr)) were included(Mainen & Sejnowski, Science 268:1503-1506, 1995) each with densities of300 pS/μm². Ca²⁺influx was mediated by a fast calcium conductance(g_(Ca); Yamada et al., Multiple channels and calcium dynamics. InMethods in Neuronal Modeling, Koch, C. & Segev, I., Eds. pp 97-134,1989) with a density of 1 pS/μm². Resultant currents were calculatedusing conventional Hodgkin-Huxley kinetic schemes according to theformulae given below. The resting membrane potential was set at −70 mVand Na and K current reversal potentials at +50 mV and −75 mV,respectively. The calcium channel was computed using theGoldman-Hodgkin-Katz equation. Extracellular Ca concentration was 2.5 mMand the intracellular Ca concentration computed using entry via I_(Ca)and removal via a first order pumpd[Ca²⁺]_(i)/dt=:(−1×10⁵.I_(Ca)/2F)−([Ca²⁺]₁−[Ca²⁺]_(∞))/τ_(R), where[Ca²⁺]_(∞)=10 mM and σ_(R) =80 ms. The time constants and maximalconductances were developed at room temperature and were thereforescaled to 37° C. using a Q₁₀ of 2.3. Formulae used for calculation ofvarious currents were as follows:

Sodium current (I _(na)), m ³ ·h: α _(m), _(Na)=0.182·(v+25)/(l−e^((v+25)/9));

β_(m), _(Na)=−0.124·(v+25)/(l−e ^(−(v+25)/9))α_(h),_(Na)=0.024·(v+40)/(1−e ^(−(v+40)/5));β_(h), _(Na)=−0.0091·(v+65)/(1−e^(−(v+65)/5));h _(∞), _(Na)=1/(1−e ^(−(v+55/6 2))

Delayed rectifier (I_(K(DR))), m: α _(m), _(K(DR))=0.02·(v−25)/(1−e^(−(v+25)/9)) ; βm, _(K(DR))=−0.002·(v −25)/(1−e ^((v+25)/9))

High threshold, N-type calcium current (I_(ca)), m·h: m _(∞),_(Ca)=1/(1+e ^(−(v−3)/8)); τ_(m), _(Ca)=7.8/(e ^((v+6)/16)); h _(Ca)=K/(K+[Ca ²⁺]_(i)) with K=0.01 mM.

[0174] The brain-dominant form, rnα_(1B-d), was then modeled by shiftingthe voltage-dependence of the N-type Ca channel conductance activationvariable (m_(∞), _(Ca)) by −7 mV, and decreasing the activation timeconstant (τ_(m), _(Ca)) by 33% (Lin et al., 1997 and see FIG. 1A).

[0175] Ribonuclease Protection Assay

[0176] The procedures are essentially the same as those described in Linet al. (1997). Total RNA was purified from various neuronal tissue ofadult rats using a guanidium thiocyanate and phenol-chloroformextraction protocol (adapted from Chomczynski & Sacchi, Anal. Biochem.162:156-159, 1987). ³²P-labeled antisense RNA probes overlapping ET (nt4379-4836) in rnα_(1B-b) and NP (nt4605-4930) in rbα_(1A) (Starr et al.,Proc. Natl. Acad. Sci. USA, 88:5621-5625, 1991) were constructed fromlinearized plasmids (pGEM-T vector) containing appropriateRT-PCR-derived sub-clones using the Maxi-script kit (Ambion). Probeswere gel purified and stored as ethanol precipitates. 1 μg of RNApurified from sympathetic or sensory ganglia or 5 μg of RNA isolatedfrom various CNS tissues were precipitated with 2×10⁵ cpm of probe andresuspended in 30 μl hybridization buffer containing: 60% formamide; 0.4M NaCl; 10 mM EDTA and 40 mM PIPES at pH 6.4. Samples were denatured at85° C. and allowed to hybridize overnight at 60° C. The samples werethen digested in a 350 μl reaction mix containing: 0.3 M NaCl, 5mM EDTA,3.5 μl of the RNase Cocktail (Ambion) and 10 mM Tris at pH 7.5, thentreated with proteinase K, extracted and precipitated with 10 g of tRNAas carrier. After resuspension in 30 μl formamide loading buffer, thesamples wee denatured and separated on a 5% polyacrylamide gel. Afterexposure to a phosphor imaging plate to quantity relative bandintensities (Fuji BAS 1000), the gel was subsequently exposed to filmwith an intensifying screen for 4-5 days at -80° C.

[0177] Site-directed Mutagenesis

[0178] A recombinant PCR-based technique was used to introduce mutations(QT, EA, AT, AA, NP) at the ET site in the IVS3-S4 linker of α_(1B-b) .A pair of primers 5′-attcttgtggtcatcgccttgag (Bup 3460; SEQ ID NO: 13)and 5′-gacaggcctccaggagcttggtg (Bdw 5623; SEQ ID NO: 14) flanked aregion of the clone that contained two restriction sites RsrII (nt3510)and BglII (nt5465) located on either side of ET (nt4674). A secondprimer pair contained the desired mutation and directly overlapped theET site (Bdwmut and Bupmut; see below). Two separate PCRs were performedwith Bup 3460 and Bdwmut, and Bupmut and Bdw 5623. The PCR product thenserved as template for a second round of PCR using Bup 3460 and Bdw 5623generating the final mutant PCR fragment that was subsequently subclonedinto rnα_(1B-b) at the Rsr II and Bgl II sites. Mutants were screened byrestriction digest and confirmed by DNA sequencing. All PCR wasperformed using Expand High Fidelity (Boehringer Mannheim). Themutagenesis primers used were as follows: ET/AT: Bupmut5′-gagattgcgGCAACGaacaacttcatc-3′: SEQ ID NO: 15 Bdwmut5′-aagttgttCGTTTCcgcaatctccg-3t; SEQ ID NO: 16 ET/QT: Bupmut5′-gagattgcgCAGACGaacaacttcatc-3′; SEQ ID NO: 17 Bdwmut5′-aagttgttCGTCTGcgcaatctccg-3′; SEQ ID NO: 18 ET/EA: Bupmut5′-gagattgcgGAAGCTaacaacttcatc-3′; SEQ ID NO: 19 Bdwmut5′-aagttgttAGCTTCcgcaatctccg-3′; SEQ ID NO: 20 ET/AA: Bupmut5′-gagattgcgGCAGCTaacaacttcatc-3′; SEQ ID NO: 21 Bdwmut5′-aagttgttAGCTGCcgcaatctccg-3′; SEQ ID NO: 22 ET/NP: Bupmut5′-gagattgcgAACCCTaacaacttcatc-3′; SEQ ID NO: 23 Bdwmut5′-aagttgttAGGGTTcgcaatctccg-3′; SEQ ID NO: 24

[0179] Genomic Analysis

[0180] The IVS3-S4 region of the rat α_(1B) and α_(1A) genes wereanalyzed by genomic PCR. Primer pairs were directed to the IVS3 and IVS4membrane spanning regions that were presumed to reside in the 5′ and 3′exons flanking the ET and NP insertions of the α_(1B) and α_(1A) genes,respectively. PCR was performed in a 50 μl reaction mix containing 250ng rat liver genomic DNA, 250 μM of each nucleotide and 0.4 μM of eachprimer. After a pre-incubation for 15 min at 92° C., 0.75 ,μl enzyme mixwas added to start the amplification. The resultant gDNA products weregel purified, cloned into pGEM-T (Promega) and sequenced. The α_(1B)primers generated two bands of ˜11 kb and ˜900 bases. The 11 kb band wasderived from the α_(1B) gene and contained the desired ET encoding exonin IVS3-S4. The 900 base product resulted from amplification of theequivalent site in the α_(1E) gene that contained a relatively short˜700 bp intron and no intervening exon. The α_(1A) primers generated asingle 9 kb PCR product that was confirmed to be derived from the α_(1A)gene by DNA sequencing (Yale University sequencing facility). Primerswere as follows: α_(1A:) Aup4737 5′-tgcctggaacatcttcgactttgtga; SEQ IDNO: 25 Adw4876 5′-cagaggagaatgcggatggtgtaacc; SEQ ID NO: 26 α_(1B:)Bup4599 5′-cagagatgcctggaacgtctttgac; SEQ ID NO: 27 Bdw47445′-ataacaagatgcggatggtgtagcc; SEQ ID NO: 28

[0181] Alternative Splicing in the Putative S3-S4 Extracellular LinkersAffects Channel Activation but not Inactivation Kinetics

[0182] In a previous study it was shown that rnα_(1B-b) (ΔSFMG/+ET) andrnα_(1B-b) (+SFMG/ΔET) N-type currents differ with respect to theiractivation kinetics when expressed in Xenopus oocytes (compare Δ/+ and+/Δ in FIG. 1A,B; see also Lin et al., 1997). Inactivation kinetics ofthe two splice variants have not, however, been compared (Lin et al.,1997). In the present study depolarizations of durations of between 26ms and 2.6 s were employed to permit the resolution of both the timecourse of Ca channel activation and inactivation. Rat N-type calciumchannel subunits (rnα_(1B-b) [Δ/+] and rnα_(1B-d) [+/Δ]) were expressedin Xenopus oocytes and resulting N-type Ca channel currents recordedusing 5 mM Ba as the charge carrier (FIG. 1). FIG. 1A shows theaveraged, normalized Ca channel current induced by the expression inXenopus oocytes of four different α_(1B) constructs. Currents wereevoked by step depolarizations to 0 mV from a holding potential of −80mV. Each trace represents the average, normalized current calculatedfrom at least 6 oocytes. SFMG-containing clones are distinguished fromSFMG-lacking clones by thin and thick lines and arrows, respectively.FIG. 1B shows a plot of average activation time constants (nat. log) atdifferent test potentials (between −20 and +10 mV) for clones +/+ (□),Δ/+ (•), +/Δ (◯) and Δ/Δ (▪). The presence of SFMG in domain IIIS3-S4did not affect the rate of channel activation. There was no significantdifference in τ_(activ) between clones +/+ and Δ/+ or between clones +/Δand Δ/Δ (p>0.1 at all potentials between −20 mV and +10 mV). Thepresence of ET in domain IVS3-S4 slowed channel activation kinetics.τ_(activ) values for clones +/+ and Δ/+ were significantly slowercompared to +/Δ and Δ/Δ, at all test potentials between −20 mV and +10mV (p<0.05).

[0183] N-type Ca channel currents evoked by depolarization to 0 mV orhigher, inactivated with a bi-exponential time course (τ_(fast) 100-150ms and τ_(slow) 700-800 ms). The inactivation time constants of thecloned channels expressed in Xenopus oocytes (rnα_(1B-b), Δ/+ andrnα_(1B-d), +/Δ) were weakly voltage-dependent consistent with studiesof native N-type Ca channels of bullfrog sympathetic neurons (Jones &Marks, 1989). The fast and slow inactivation time constants ofrnα_(1B-b) and rnα_(1B-d) currents evoked by step depolarizations tobetween 0 mV and +mV were not significantly different. In contrast, therates of channel activation of the two variants in the same cells weresignificantly different (FIG. 1 A,B). On the basis of these observationsit was concluded that alternative splicing in domains IIIS3-S4 andIVS3-S4 of the α_(1B)-subunit altered the time course of N-type Cachannel activation but had no effect on inactivation kinetics. Thesefindings are consistent with the close proximity of the S3-S4 linkers totheir respective S4 helices that are the putative voltage sensors of the6 transmembrane family of voltage-gated ion channels. In contrast, thedomains of the Ca channels α_(1B) subunit implicated involtage-dependent inactivation of N-type Ca channels (IS6 and flankingputative extracellular and intracellular linkers; Zhang et al., Nature372:97-100, 1994) are likely to be more distant from the S3-S4 linkersplice sites.

[0184] The Observed Differences in the Properties of rnα_(1B-b) andrnα_(1B-d) Currents are of Sufficient Magnitude to Impact ActionPotential-Induced Ca Entry

[0185] An assumption that motivates the present study is that thedifferences in the kinetics and voltage-dependence of activation ofrnα_(1B-b) and rnα_(1B-d) N-type Ca channel currents are sufficient toinfluence the magnitude and time course of voltage-dependent calciumentry in native cells. A direct test of this hypothesis, however, iscomplicated by the inability to manipulate selectively the expression oractivity of individual splice variants in their native environment. Todate no isoform-specific pharmacological tools or antibodies to targetCa channel α_(1B) S3-S4 splice variants exist. Therefore, the availableinformation was used to estimate the relative effectiveness ofrnα_(1B-b) and rnα_(1B-d) N-type currents to support actionpotential-induced Ca influx in a model neuron (Hines & Carnevale, 1997).A one-compartment model was used to predict the time course andmagnitude of calcium entry in a neuron during action potential-induceddepolarization. Simulated action potentials with time courses similar tothose recorded in native sympathetic neurons (Yamada et al., 1989; FIG.2A) were used to trigger voltage-dependent Ca influx in model neurons(Na, K and Ca current densities of 300, 300 and 1 pS/μF, respectively)expressing either rnα_(1B-b) or rnα_(1B-d) N-type Ca channel currents. Asimulated action potential was evoked by a 10 ms, 40 pA current step(FIG. 2A); a comparison of the resultant N-type channel current (FIG.2B) and time course of intracellular calcium concentration (FIG. 2C)expected in a model neuron expressing either rnα_(1B-b) (Δ/+; solidline) or rnα_(1B-d) (+/Δ; dashed line)-type channels is shown. A shiftin the voltage-dependence of the N-type Ca channel conductanceactivation variable (m_(∞), _(Ca)) by −7 mV, and a decrease in theactivation time constrant (τ_(m), _(Ca)) by 33% expected for rnα_(1B-d)(Lin et al., 1997; and see FIG. 1A), resulted in a total increase incharge transfer and peak intracellular Ca concentration of 49% and 48%,respectively. A −50% increase in the total charge transfer (FIG. 2B) andpeak intracellular Ca concentration (FIG. 2C) is predicted during anaction potential in a neuron expressing rnα_(1B-d)-type Ca channels(dashed line) relative to rnα_(1B-b) (solid line). All other factorsbeing constant, the functional differences between rnα_(1B-b) andrnα_(1B-d) N-type Ca channel currents would be expected to significantlyimpact the amount of calcium that enters a neuron during actionpotential-dependent excitation.

[0186] Splicing of ET in Domain IVS3-S4 Underlies the Major FunctionalDifference Between rnα_(1B-b) and rnα_(1B-d)

[0187] rnα_(1B-b) and rnα_(1B-d) differ in composition by 6 amino acidslocated in two distinct regions of the Ca channel α_(1B) subunit (SFMGin domain IIIS3-S4 and ET in domain IVS3-S4). To separate the relativecontribution of SFMG in domain IIIS3-S4 and ET in domain IVS3-S4 to thedifferent gating kinetics observed between rnα_(1B-b) (ΔSFMG/+ET) andrnα_(1B-d) (+SFMG/ΔET) two additional clones, +/+ and Δ/Δ wereconstructed and the functional properties of all four clones werecompared. FIG. 1 (A and B) demonstrates that the presence of thedipeptide sequence ET in domain IVS3-S4 is directly correlated with thealtered activation kinetics of rnα_(1B-b) currents compared tornα_(1B-d). Activation time constants measured from N-type Ca channelcurrents in oocytes expressing clone Δ/+ (rnα_(1B-b)) and +/+ wereindistinguishable and 1.5 fold slower on average than those induced bythe expression of clones +/Δ (rnα_(1B-d)) and Δ/Δ (FIG. 1A,B). Thepresence of ET in domain IVS3-S4 also influenced the voltage-dependenceof channel activation. A comparison of the mid-points of the risingphase of the peak current-voltage plots (V_(½)) generated for the two ETcontaining clones, Δ/+ (rnα_(1B); −7.8 ±0.6 mV, n=6) and +/+ (−9.7 ±1.0mV, n=6) shows that they are not significantly different from each other(p>0.05, students' t-test). Likewise, V_(½) values estimated from twoET-lacking constructs, +/Δ (rnα_(1B-d);−15.4±0.4 mV, n=7) and Δ/Δ(−13.4±0.7, n=6), were not significantly different from each other(p>0.05) and activated at potentials that were, on average, 6 mV morenegative compared to ET-containing clones Δ/+ and +/+. While thepresence of ET in domain IVS3-S4 dominates in regulating thevoltage-dependence of activation, the analysis does reveal a smallcontribution of SFMG. SFMG-containing clones (+/Δ and +/+) activated atpotentials that were 2 mV hyperpolarized compared to those that lackedSFMG (Δ/+ and Δ/Δ). A 2 mV shift in the voltage-dependence of activationwas not significant at the 5% level, in a comparison of V_(½) valuesfrom clones Δ/+ and +/+, but did reach significance in a comparison of+/Δ and Δ/Δ (p<0.025, students t-test).

[0188] The Pattern of Expression of ET-Containing Ca Channel α_(1B) mRNAin Different Regions of the Nervous System

[0189]FIG. 1 indicates that alternative splicing of ET within domainIVS3-S4 of the Ca channel α_(1B)-subunit accounts for the majorfunctional differences between rnα_(1B-b) and rnα_(1B-A). This prompteda systemic analysis of the expression pattern of the six bases in α_(1B)mRNA that encoded ET (gaa acg). It was previously shown thatET-containing α_(1B) (+ET α_(1B)) mRNA was in very low abundance intotal rat brain extracts (Lin et al., 1997). To determine whetherET-lacking α_(1B) (ΔET α_(1B)) mRNA dominated throughout the centralnervous system RNA isolated from spinal cord, cerebellum, cortex,hippocampus, hypothalamus, medulla and thalamus of adult rats wasanalyzed by ribonuclease protection assay. In all regions tested >90% ofthe U,B mRNA expressed in the central nervous system lacked the ETencoding sequence. In contrast, in sympathetic and sensory ganglia themajority of α_(1B) mRNA contained the ET encoding sequence. Togetherthese findings suggest that +ET α_(1B) subunits are primarily restrictedto neurons of the peripheral nervous system. Consistent with this RNAisolated from human brain and trigeminal ganglia was analyzed andanalogous patterns of expression were observed: low levels of +ET α_(1B)mRNA in brain and high levels (>90%) in ganglia.

[0190] Site-Directed Mutagenesis Within IVS3-S4

[0191] Having shown that alternative splicing of the ET encodingsequence in the IVS3-S4 linker of α_(1B) has a significant effect on thekinetics and voltage-dependence of N-type Ca channel gating, the use ofsite-directed mutagenesis was employed to determine the relativeimportance of each amino acid, glutamate and threonine. A series ofmutants in which ET was replaced with either QT, AT, EA, AA or NP wereconstructed (FIG. 3) from clone Δ/+ (rn α_(1B-b)) which served as thebackground structure. The mutant constructs were then expressed inXenopus oocytes and their properties compared to clones +ET (100% slow;FIG. 3) and ΔET (100% fast; FIG. 3). All mutants expressed equally wellin the Xenopus oocyte expression system.

[0192] The role of the glutamate in domain IVS3-S4 was of major interestbecause it should be negatively charged at neutral pH and consequentlymight influence the gating machinery of the channel via electrostaticinteractions. FIG. 3, however, shows that replacing glutamate withglutamine resulted in a channel that activated only slightly faster than+ET α_(1B) (FIG. 3; QT). Substituting alanine for glutamate (AT)decreased τ_(act) but, consistent with the QT mutant, suggests that thepresence of a negative charge in IVS3-S4 (glu) does not underlie theslow gating kinetics of the +ET α_(1B) variant. Similarly, alaninesubstitution of either threonine alone (EA) or together with glutamate(AA) generated channels with activation kinetics that were intermediatebetween +ET α_(1B) and ΔET α_(1B) clones. Together, these resultssuggest that the presence of both glutamate and threonine in the IVS3-S4linker is necessary to reconstitute the relatively slow channel openingrates characteristic of N-type Ca channel α_(1B)-subunits that dominatein sensory and sympathetic ganglia.

[0193] Sequence comparisons of several cDNAs encoding α₁-subunits ofother voltage-gated Ca channels suggests that alternative splicing inthe IVS3-S4 linker could be a general mechanism for regulatingvoltage-dependent Ca channel gating. This has recently been demonstratedfor α_(1A) (Sutton et al., Soc. Neurosci. Abs. 24:21, 1998), a Cachannel subunit that is closely related both structurally andfunctionally to the N-type Ca channel α_(1B) subunit. A comparison ofthe IVS3-S4 region of various mammalian α_(1A) cDNAs derived fromkidney, pancreas and brain (see also Yu et al., Proc. Natl. Acad. Sci.USA 89:10494-10498, 1992; Ligon et al., J Biol. Chem. 273:13905-13911,1998; Sutton et al., 1998) is consistent with alternative splicing ofsix bases encoding Asp Pro (NP) amino acids in this region. Thedistribution of +NP α_(1A) and ΔNP α_(1A) mRNAs in different regions ofthe rat nervous system has not been quantified. Therefore RNaseprotection analysis was used to determine the expression pattern of theIVS3-S4 splice variants of α_(1A). Low levels of +NP α_(1A) mRNA werefound in rat, spinal cord, striatum and thalamus, a pattern thatparallels the low levels of +ET α_(1B) mRNA in the CNS. However, thepattern of NP expression in the cerebellum, cortex and hippocampus didnot conform to this picture since mRNA isolated from these tissuescontained a significant proportion of +NP α_(1A) mRNAs. In fact, in thehippocampus +NP α_(1A) mRNAs dominated (˜60%). Consistent with theabundance of +ET α_(1B) mRNAs in peripheral tissue, the majority ofα_(1A) mRNA in superior cervical and dorsal root ganglia contained thesix bases encoding NP in domain IVS3-S4 of α_(1A). The absolute level ofα_(1A) mRNA expressed in sympathetic neurons was very low as expectedfrom the absence of P-type currents in recordings from rat sympatheticneurons (Mintz et al., 1992).

[0194] The high degree of sequence homology between α_(1B) and α_(1A) inthe IVS3-S4 linker region together with the finding that a 6 basesequence is alternatively spliced at both these sites, suggested that ETand NP share a common functional role. To test this hypothesis thefunctional impact on N-type Ca channel currents of replacing ET inrnα_(1B-b) with NP was studied. FIG. 3 shows that the +NP α_(1B) mutantgives rise to N-type Ca channel currents in oocytes with gating kineticsindistinguishable from wild-type (i.e. +ET α_(1B))Activation timeconstants were estimated from currents induced by the expression of thevarious mutant α_(1B) constructs (QT, AT, EA, AA, NP) in oocytes andcompared to clones ET and ΔET (A). Shifts in the activation timeconstants of the mutant channels, relative to clones ET and ΔET (100%slow) and ΔET (100% fast) are plotted (B). Each point represents datacollected from at least 18 oocytes per mutant (each mutant was tested inthree separate batches of oocytes and within each experiment at least 6oocytes per mutant were analyzed). Values plotted are means ±standarderrors from the three data sets. The asterisk indicates a significantslowing of the activation time constant compared to clone ET (P<0.05).

[0195] ET is Encoded by a Six Base Exon in the IVS3-S4 Linker Region ofthe α_(1B) Gene

[0196] The existence of an alternatively spliced exon in the IVS3-S4region of the rat Ca channel α_(1B) gene has been hypothesized (Lin etal., 1997), but not yet confirmed. Genomic analysis was thereforeundertaken to locate the splice junctions in the IVS3-S4 region of theα_(1B) gene and to pinpoint the precise location of the putativesix-base, ET encoding exon. PCR amplification from rat genomic DNA usingprimers designed to hybridize to the transmembrane spanning S3 and S4helices flanking IVS3-S4 in α_(1B) revealed the presence of a long ˜10kb stretch of intron sequence. DNA sequencing established the locationof exon/intron and intron/exon boundaries and conserved ag-gt splicejunction signature sequences immediately 5′ and 3′ to the putative ETinsertion site. A six-base cassette exon encoding ET was located 8 kbinto the 5′ intron and establishes that ET-α_(1B) variants are generatedby alternative splicing. The exon/intron structure in the IVS3-S4 linkerregion of the closely related rat α_(1A) gene was also determined. Therat α_(1A) gene also contained a long stretch of intron sequence (˜8 kb)and ag-gt splice junctions at the 5′ (gt) and 3′ (at) ends of theintronic segment. The precise location of the NP encoding cassette exonin the rat α_(1A) gene has not been determined but conclude that it mustreside within the 8 kb of intron sequence in the IVS3-S4 linker region.Tissue-specific alternative splicing of six base cassette exons in theIVS3-S4 linkers of both α_(1A) and α_(1B) explains the presence ofsplice variants of these subunits in the mammalian brain and underscoresthe high level of conservation between these two functionally relatedgenes. The genomic structure of the more distantly related rat α_(1E)gene that encodes a pharmacologically and functionally distinct class ofCa channel (Soong et al., Science 260:1133-1136, 1993) also wasanalyzed. The α_(1E) gene contains a ˜700 bp intron in the IVS3-S4linker region and no obvious intervening exon. The absence of analternatively spliced cassette exon in the IVS3-S4 linker region of theα_(1E) gene is consistent with RNase protection analysis of α_(1E) mRNAfrom rat brain which revealed no evidence of sequence variations in thisIVS3-S4 linker.

[0197] Each of the foregoing patents, patent applications and referencesis hereby incorporated by reference. While the invention has beendescribed with respect to certain embodiments, it should be appreciatedthat many modifications and changes may be made by those of ordinaryskill in the art without departing from the spirit of the invention. Itis intended that such modification, changes and equivalents fall withinthe scope of the following claims.

What is claimed is:
 1. A human N-type calcium channel hα_(1B+SFVG)subunit polypeptide which comprises the amino acid sequence of SEQ IDNO:
 2. 2. The human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:
 4. 3. The human N-type calcium channelhα_(1B+SFVG) subunit polypeptide of claim 1, wherein the polypeptideconsists of the amino acid sequence of SEQ ID NO:
 4. 4. A human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide comprising a fragmentor variant of the polypeptide of claim 1, wherein the fragment orvariant comprises the amino acid sequence of SEQ ID NO:
 2. 5. A humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid molecule whichencodes the polypeptide of any of claims 1-4.
 6. The human N-typecalcium channel hα_(1B+SFVG) subunit nucleic acid molecule of claim 5,wherein the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid molecule comprises the nucleotide sequence of SEQ ID NO:
 1. 7. Thehuman N-type calcium channel hα_(1B+SFVG) subunit nucleic acid moleculeof claim 5, wherein the human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid molecule comprises the nucleotide sequence of SEQID NO:
 3. 8. The human N-type calcium channel hα_(1B+SFVG) subunitnucleic acid molecule of claim 5, wherein the human N-type calciumchannel hα_(1B+SFVG) subunit nucleic acid molecule consists of thenucleotide sequence of SEQ ID NO:
 3. 9. The human N-type calcium channelhα_(1B+SFVG) subunit nucleic acid molecule of claim 8, wherein the humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid is a homolog orallele of the nucleic acid sequence of SEQ ID NO:
 3. 10. A fragment ofthe human N-type calcium channel hα_(1B+SFVG) subunit nucleic acidmolecule of claim
 5. 11. An expression vector comprising the humanN-type calcium channel hα_(1B+SFVG) subunit nucleic acid molecule ofclaim 5 operably linked to a promoter.
 12. An expression vectorcomprising the human N-type calcium channel hα_(1B+SFVG) subunit nucleicacid molecule of claim 10 operably linked to a promoter.
 13. A host celltransformed or transfected with the expression vector of claim
 11. 14.An agent which selectively binds the human N-type calcium channelhα_(1B+SFVG) subunit polypeptide of claim 1 or a nucleic acid thatencodes the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide of claim
 1. 15. The agent of claim 14, wherein the agent isa polypeptide which binds selectively to the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide.
 16. The agent of claim 15,wherein the polypeptide is a monoclonal antibody or a polyclonalantibody.
 17. The agent of claim 15, wherein the polypeptide is anantibody fragment selected from the group consisting of a Fab fragment,a F(ab)₂ fragment and a fragment including a CDR3 region.
 18. The agentof claim 14, wherein the agent is an antisense nucleic acid whichselectively binds to a nucleic acid encoding the human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide.
 19. The agent of claims 14-18,wherein the agent is an inhibitor of the calcium channel activity of thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide.
 20. Acomposition comprising a pharmaceutically acceptable carrier and acomponent selected from the group consisting of the polypeptide of claim1, the nucleic acid molecule of claim 5 and the agent of claim
 14. 21. Amethod for inhibiting human N-type calcium channel hα_(1B+SFVG) subunitactivity in a mammalian cell comprising contacting the mammalian cellwith an amount of a human N-type calcium channel hα_(1B+SFVG) subunitinhibitor effective to inhibit calcium influx in the mammalian cell. 22.The method of claim 21, wherein the inhibitor is selected from the groupconsisting of an antibody which selectively binds the human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide, an antisense nucleicacid which binds a nucleic acid encoding human N-type calcium channelhα_(1B+SFVG) subunit polypeptide and a dominant negative human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide.
 23. A method fortreating a subject having a stroke, neuropathic pain, or traumatic braininjury comprising administering to a subject in need of such treatmentan inhibitor of the human N-type calcium channel hα_(1B+SFVG) subunitpolypeptide in an amount effective to inhibit voltage regulated calciuminflux.
 24. The method of claim 23, wherein the inhibitor is selectedfrom the group consisting of an antibody which selectively binds thehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide, anantisense nucleic acid which binds a nucleic acid encoding human N-typecalcium channel hα_(1B+SFVG) subunit polypeptide and a dominant negativehuman N-type calcium channel hα_(1B+SFVG) subunit polypeptide.
 25. Themethod of claim 23, wherein the inhibitor is administeredprophylactically to a subject at risk of having a stroke.
 26. A methodfor increasing human N-type calcium channel hα_(1B+SFVG) subunitexpression in a cell comprising contacting the cell with a moleculeselected from the group consisting of a human N-type calcium channelhα_(1B+SFVG) subunit nucleic acid and a human N-type calcium channelhα_(1B+SFVG) subunit polypeptide, in an amount effective to increasevoltage regulated calcium influx in the cell.
 27. The method of claim26, wherein the cell is contacted with one or more human N-type calciumchannel non-hα_(1B+SFVG) subunits of the human N-type calcium channel ornucleic acids encoding such subunits.
 28. A method for increasingcalcium channel voltage regulated calcium influx in a subject comprisingadministering to a subject in need of such treatment a molecule selectedfrom the group consisting of a human N-type calcium channel hα_(1B+SFVG)subunit nucleic acid and a human N-type calcium channel hα_(1B+SFVG)subunit polypeptide in an amount effective to increase voltage regulatedcalcium influx in the subject.
 29. A method for identifying leadcompounds for a pharmacological agent useful in the treatment of diseaseassociated with increased or decreased voltage regulated calcium influxmediated by a human N-type calcium channel comprising providing a cellor other membrane-encapsulated space comprising a human N-type calciumchannel hα_(1B+SFVG) subunit polypeptide; contacting the cell or othermembrane-encapsulated space with a candidate pharmacological agent underconditions which, in the absence of the candidate pharmacological agent,cause a first amount of voltage regulated calcium influx into the cellor other membrane-encapsulated space; determining a test amount ofvoltage regulated calcium influx as a measure of the effect of the leadcompounds for a pharmacological agent on the voltage regulated calciuminflux mediated by a human N-type calcium channel, wherein a the testamount of voltage regulated calcium influx which is less than the firstamount indicates that the candidate pharmacological agent is a leadcompound for a pharmacological agent which reduces voltage regulatedcalcium influx and wherein a test amount of voltage regulated calciuminflux which is greater than the first amount indicates that thecandidate pharmacological agent is a lead compound for a pharmacologicalagent which increases voltage regulated calcium influx.
 30. The methodof claim 29, further comprising the step of loading the cell or othermembrane-encapsulated space with a calcium-sensitive compound which isdetectable in the presence of calcium, wherein the calcium-sensitivecompound is detected as a measure of the voltage regulated calciuminflux.
 31. A method for identifying compounds which selectively bind ahuman N-type calcium channel hα_(1B+SFVG) subunit isoform comprising,providing a first cell or membrane encapsulated space which expresses ahuman N-type calcium channel hα_(1B+SFVG) subunit isoform, providing asecond cell or membrane encapsulated space which expresses a humanN-type calcium channel non-hα_(1B+SFVG) subunit isoform, wherein thesecond cell or membrane encapsulated space is identical to the firstcell except for the α_(1B) isoform expressed, contacting the first cellor membrane encapsulated space and the second cell or membraneencapsulated space with a compound, determining the binding of thecompound to the first cell or membrane encapsulated space and the secondcell or membrane encapsulated space, wherein a compound which binds thefirst cell or membrane encapsulated space but does not bind the secondcell or membrane encapsulated space is a compound which selectivelybinds the human N-type calcium channel hα_(1B+SFVG) subunit isoform. 32.A method for identifying compounds which selectively bind a human N-typecalcium channel hα_(1B+SFVG) subunit isoform comprising, providing ahuman N-type calcium channel hα_(1B+SFVG) subunit isoform polypeptide ornucleic acid, providing a human N-type calcium channel non-hα_(1B+SFVG)subunit isoform polypeptide or nucleic acid, contacting the human N-typecalcium channel hα_(1B+SFVG) subunit isoform polypeptide or nucleic acidand the human N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid with a compound, determining the binding ofthe compound to the human N-type calcium channel hα_(1B+SFVG) subunitisoform polypeptide or nucleic acid and the human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid, wherein acompound which binds the human N-type calcium channel hα_(1B+SFVG)subunit isoform polypeptide or nucleic acid but does not bind the humanN-type calcium channel non-hα_(1B+SFVG) subunit isoform polypeptide ornucleic acid is a compound which selectively binds the human N-typecalcium channel hα_(1B+SFVG) subunit isoform.
 33. A method foridentifying compounds which preferentially bind a human N-type calciumchannel hα_(1B+SFVG) subunit isoform comprising, providing a first cellor membrane encapsulated space which expresses a human N-type calciumchannel hα_(1B+SFVG) subunit isoform, providing a second cell ormembrane encapsulated space which expresses a human N-type calciumchannel non-hα_(1B+SFVG) subunit isoform, wherein the second cell ormembrane encapsulated space is identical to the first cell except forthe α_(1B) isoform expressed, contacting the first cell or membraneencapsulated space and the second cell or membrane encapsulated spacewith a compound, determining the binding of the compound to the firstcell or membrane encapsulated space and the second cell or membraneencapsulated space, wherein a compound which binds the first cell ormembrane encapsulated space in an amount greater than the compound bindsthe second cell or membrane encapsulated space is a compound whichpreferentially binds the human N-type calcium channel hα_(1B+SFVG)subunit isoform.
 34. A method for identifying compounds whichpreferentially bind a human N-type calcium channel hα_(1B+SFVG) subunitisoform comprising, providing a human N-type calcium channelhα_(1B+SFVG) subunit isoform polypeptide or nucleic acid, providing ahuman N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid, contacting the human N-type calcium channelhoc hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid and thehuman N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid with a compound, determining the binding ofthe compound to the human N-type calcium channel hα_(1B+SFVG) subunitisoform polypeptide or nucleic acid and the human N-type calcium channelnon-hα_(1B+SFVG) subunit isoform polypeptide or nucleic acid, wherein acompound which binds the human N-type calcium channel hα_(1B+SFVG)subunit isoform polypeptide or nucleic acid in an amount greater thanthe human N-type calcium channel non-hα_(1B+SFVG) subunit isoformpolypeptide or nucleic acid is a compound which preferentially binds thehuman N-type calcium channel hα_(1B+SFVG) subunit isoform.
 35. A methodfor selectively treating subject having a condition characterized byaberrant brain neuronal calcium current comprising, administering to asubject in need of such treatment a pharmacological agent which isselective for a human N-type calcium channel hα_(1B+SFVG) subunit, in anamount effective to normalize the aberrant neuronal calcium current.